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Abstract Eusociality in bees represents a major evolutionary transition and understanding its molecular basis is fundamental for sociogenomic studies. Comparative genomics has revealed correlations between transcription factor binding site (TFBS) abundance and social complexity; however, when and where these TFBSs function in a eusocial context remains largely unclear. In this study, we performed cap analysis of gene expression (CAGE) during worker metamorphosis in the honeybee Apis mellifera to identify TFBSs within active enhancers and decipher the regulatory relationships between these enhancers and their target genes. We identified 17,349 transcription start sites (TSSs) and 842 candidate enhancers. Using CAGE, we identified five clusters based on expression patterns. Notably, genes associated with the canonical metamorphic regulators, Broad complex ( Br-c ) and E93 , were found within specific clusters. By integrating the correlations between enhancer and TSS activities with motif enrichment analysis, we identified 15 transcription factor–enhancer–TSS regulatory relationships. Among these, tramtrack ( ttk )-binding sites were identified in five enhancers associated with four target genes, including Br-c . The number of target genes regulated by ttk was the highest in our dataset. To examine whether this regulatory relationship is conserved across bee species with varying levels of sociality, we analyzed the sequence conservation of ttk -binding sites in Br-c enhancers and found that perfect sequence conservation of ttk -binding site was restricted to the Apis genus. The ttk -binding sites of other target genes exhibited the same Apis -specific conservation pattern. Our findings suggest that gene regulatory relationships during worker metamorphosis occur in a lineage-specific manner in the Apis genus. Significance Honeybees produce distinct castes—queens and workers—from genetically identical larvae via differences in gene regulation. Although enhancers have been computationally predicted, their actual activity during bee development has rarely been measured directly, and the CAGE technology has never been applied for this purpose. We identified active enhancers during worker metamorphosis and discovered that the transcription factor ttk may regulate Br-c , a key developmental gene. This study provides the first direct evidence of active enhancers and their regulatory roles in honeybee worker metamorphosis.