MicroRNAs play an important role in the adipogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). How miR-100-3p influences such adipogenesis, however, remains uncertain. In this study, hMSC adipogenic differentiation was associated with miR-100-3p downregulation, and overexpressing this miRNA inhibited adipogenesis and the expression of adipogenic marker genes. Through bioinformatics approaches, miR-100-3p can bind the 3'-untranslated region (3′-UTR) of the mRNA encoding phosphoinositide 3-kinase regulatory subunit 1 (PIK3R1) such that miR-100-3p overexpression resulted in significant reductions in PIK3R1 expression. Importantly, overexpressing PIK3R1 was sufficient to reverse the anti-adipogenic effects of miR-100-3p overexpression. PIK3R1 is a critical component of the PI3K/AKT signaling pathway, and miR-100-3p overexpression resulted in reduced AKT phosphorylation in the context of adipogenesis. In addition, the adipogenic differentiation of hMSCs in which miR-100-3p was overexpressed was further enhanced upon treatment with the PI3K/AKT agonist 740Y-P relative to miR-100-3p overexpression alone. Taken together, these findings provide evidence that miR-100-3p inhibits the adipogenic differentiation of hMSCs by targeting PIK3R1 via the PI3K/AKT signaling pathway.