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The 14-3-3 proteins are emerging as important modulators of osteoblast differentiation and function. Recent studies highlight specific roles of 14-3-3 paralogs in bone physiology, with their dysregulation linked to impaired skeletal homeostasis and bone-related diseases. Among these, the 14-3-3γ paralog has been implicated in bone formation, though its precise role remains unclear.In this study, we investigated the function of 14-3-3γ in the osteogenic differentiation of human adipose-derived mesenchymal stem/stromal cells (hASCs). Using an adenoviral system, we knocked down 14-3-3γ and assessed osteogenic markers. Tissue-Nonspecific Alkaline Phosphatase (TNAP) activity, RUNX2 protein levels, and the expression of osteogenic genes (BGLAP, SPP1) were analyzed during matrix maturation and mineralization. Calcium and collagen deposition were evaluated via Alizarin Red S and Aniline Blue staining, respectively, and compared with cells overexpressing recombinant 14-3-3γ. Proteomic profiling via quantitative mass spectrometry was performed to identify protein changes after 14-3-3γ silencing. Subcellular localization of endogenous 14-3-3γ was also examined during differentiation. Knockdown of 14-3-3γ enhanced TNAP activity and increased matrix mineralization, while its overexpression suppressed these processes. Proteomic analysis revealed enrichment of proteins related to endoplasmic reticulum stress and bone development. Furthermore, 14-3-3γ shifted from a diffuse to a peri-endoplasmic reticulum distribution, with increased colocalization with calnexin during osteogenic induction. These findings reveal a novel inhibitory role of 14-3-3γ in matrix mineralization of hASCs, suggesting that targeting this paralog may offer new avenues for therapies in bone remodeling disorders.