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Fat deposition plays a crucial role in regulating the production performance and meat quality of broilers. Although the heterogeneity of mammalian adipocytes has been extensively studied, research on the molecular mechanisms underlying differences in lipid droplet accumulation in avian adipocytes remains limited. This study confirmed a significant positive correlation (R<sup>2</sup> > 0.81, <i>p</i> < 0.001) between the SSC signal and lipid droplet content via fluorescence staining of lipid droplets, Oil Red O staining, and triglyceride (TG) quantification. Based on this, a label-free sorting strategy using SSC signals was established to sort differentiated chicken preadipocytes, obtaining high lipid droplet (H) and low lipid droplet (L) subpopulations, which were subsequently subjected to transcriptome sequencing and differential gene expression (DEG) analysis, followed by GO and KEGG enrichment analysis. The results indicated no significant differences in the expression of adipogenesis marker genes (<i>PPARG</i>, <i>LPL</i>, <i>CD36</i>, <i>PLIN1</i>, <i>PLIN2</i>) between the high lipid droplet (H) and low lipid droplet (L) groups, suggesting that both groups are at similar stages of differentiation. KEGG analysis revealed that both the H vs. NC and L vs. NC comparisons were enriched in common pathways, including the PPAR signaling pathway, ECM-receptor interaction, focal adhesion, cytokine-receptor interaction, and calcium-Apelin signaling pathway, suggesting that both groups of cells had activated the adipogenesis program. GO analysis showed that, in both H vs. NC and L vs. NC comparisons, differentially expressed genes (DEGs) were enriched in biological processes (BPs) related to cell adhesion, nucleosome assembly, chromatin remodeling, and receptor activity, as well as cellular components (CCs) such as the extracellular matrix, cytoskeleton, and nucleosome organization, indicating extensive gene reprogramming and activation of signaling transduction during differentiation. In the H vs. L comparison, enriched pathways included ABC transporters, ECM-receptor interaction, focal adhesion, gap junctions, microtubule-related processes, and neuroactive ligand-receptor interactions, involving lipid transmembrane transport, cytoskeleton stabilization, and signal transduction regulation, suggesting that high lipid droplet cells are more mature in lipid droplet transport, storage, and homeostasis maintenance. GO enrichment results further supported this conclusion, as H vs. L specifically enriched processes related to microtubule-related processes, cell cycle, and redox reactions (BPs), as well as chromosome organization, cytoskeleton, and motor activity (CC/MF), indicating that high lipid droplet cells maintain lipid droplet fusion and metabolic homeostasis via enhanced microtubule transport and antioxidant regulation. Differential gene analysis revealed that the L group upregulated genes associated with fatty acid synthesis and elongation (<i>ACACA</i>, <i>FASN</i>, <i>SCD</i>, <i>FADS2</i>, <i>ELOVL1</i>), cholesterol and isoprenoid biosynthesis (<i>HMGCR</i>, <i>SQLE</i>, <i>MSMO1</i>, <i>DHCR7</i>, <i>DHCR24</i>, <i>FDPS</i>, <i>LSS</i>), and fatty acid oxidation (<i>PPARA</i>, <i>PPARD</i>, <i>ACAD11</i>, <i>SIRT5</i>), reflecting a metabolic characteristic of concurrent lipid synthesis and mobilization; the H group, conversely, upregulated genes associated with lipid droplet formation and storage (<i>G0S2</i>, <i>MOGAT1</i>, <i>GPAT4</i>, <i>PLIN4</i>, <i>AUP1</i>), lipid transport (<i>ABCA1</i>, <i>ABCA2</i>, <i>ABCG1</i>, <i>OSBPL3</i>, <i>VLDLR</i>), and antioxidant defense (<i>GPX3</i>, <i>GPX4</i>, <i>HMOX1</i>), exhibiting a storage and homeostasis-oriented metabolic state. In the NC, L, and H groups, the expression of five genes-<i>GEM</i>, <i>SPP1</i>, <i>ABCA1</i>, <i>PDLIM3</i>, and <i>ITGA8</i>-showed a gradual increase, suggesting that these genes were associated with preadipocyte differentiation and lipid droplet deposition. In summary, although the high and low lipid droplet subpopulations of chicken preadipocytes exhibit similar differentiation states, they form distinct metabolic orientations. The L group is characterized by active lipid synthesis, fatty acid oxidation, and membrane lipid remodeling, while the H group predominantly features lipid droplet storage, lipid transport, and antioxidant homeostasis. This study highlights the molecular mechanisms underlying the metabolic heterogeneity of avian adipocytes and provides a theoretical basis for poultry fat deposition regulation and genetic improvement.