A high incidence of decreased bone mineral density (BMD) has increasingly been associated with HIV infection. In this study mesenchymal stem cell (MSC) and human osteoblast (hOB) cell lines were treated with HIV tat, HIV rev, HIV p55-gag, HIV gp120 and HTLV env (100 ng/ml, 24 h). Cells were then analyzed for calcium deposition, alkaline phosphatase (ALP) activity, and lipid levels using established methods. Real-time PCR with gene-specific primers was used to quantify the mRNA levels of the transcription factors RUNX-2 and PPARgamma, transcription factors known to be pro-osteogenic and pro-adipogenic, respectively. The levels of secreted bone markers and transcription factor activity were determined using commercial assays. In OBs, HIV p55-gag and gp120 were seen to reduce calcium deposition, ALP activity, levels of secreted BMP-2, -7, and RANK-L, and the expression and activity of RUNX-2. The levels of osteocalcin were also significantly reduced by p55-gag treatment, while gp120 also increased PPARgamma activity. Lipid levels were also increased by gp120 treatment. The ability of MSCs to develop into functioning OBs was also affected by the presence of HIV proteins, with p55-gag inducing a decrease in osteogenesis, while rev induced an increase. HIV proteins can potentially modulate OB development and function in vitro via modulation of bone maker secretion and RUNX-2 and PPARgamma transcription factor activity.