Previous studies of global binding patterns identified the epigenetic factor, EZH2, as a regulator of the homeodomain-only protein homeobox (HOPX) gene expression during bone marrow stromal cell (BMSC) differentiation, suggesting a potential role for HOPX in regulating BMSC lineage specification. In the present study, we confirmed that EZH2 direct binds to the HOPX promoter region, during normal growth and osteogenic differentiation but not under adipogenic inductive conditions. HOPX gene knockdown and overexpression studies demonstrated that HOPX is a promoter of BMSC proliferation and an inhibitor of adipogenesis. However, functional studies failed to observe any affect by HOPX on BMSC osteogenic differentiation. RNA-seq analysis of HOPX overexpressing BMSC during adipogenesis, found HOPX function to be acting through suppression of adipogenic pathways associated genes such as ADIPOQ, FABP4, PLIN1 and PLIN4. These findings suggest that HOPX gene target pathways are critical factors in the regulation of fat metabolism.