Osteoporosis (OP) is considered a complex disease with a strong genetic impact, mainly affecting post-menopausal women and is also a common cause of fracture. Elucidating the molecular mechanisms that regulate the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) is crucial to developing treatment strategies to combat OP. In the present study, we found that ectopic viral integration site‑1 (Evi1) was highly expressed during the process of adipogenesis of rat BMSCs. Notably, Evi1 levels markedly increased on day 3 of adipogenic differentiation following the addition of adipogenic induction supplements. In addition, we interfered with the expression of the Evi1 gene in the adipogenesis of BMSCs by supplementing adenoviral plasmids and measured the expression levels of bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN), peroxisome proliferator‑activated receptor γ2 (PPARγ2) and lipoprotein lipase (LPL) by RT-qPCR and western blot analysis. The mRNA and protein levels of osteogenic and adipogenic markers in the BMSCs were up‑ and downregulated, respectively following the silencing of siEvi1. Our experimental results substantiate that the suppression of Evi1 in BMSCs by RNA interference inhibits adipogenic differentiation, while it promotes osteogenic differentiation. The results from our study demonstrated that the Evi1 gene may be targeted as a therapeutic strategy for promoting bone formation.