Sodium butyrate (SB) has been shown to affect the differentiation of mesenchymal stem cells (MSC) through the activation of different transcriptional pathways. The aim of this study was to investigate the effects of SB on the proliferation and differentiation potential of porcine bone marrow-derived MSC. Third passage MSC were verified by flow cytometry to be > 95% CD105⁺, CD90⁺, CD44⁺, CD45⁻, and CD34⁻. Cells were cultured in either a low glucose DMEM+10% FBS (BM), BM + 2.5 mM SB (SB2.5) or BM + 5 mM SB (SB5) in a randomized complete block design. Data were analyzed with 1-way ANOVA followed by linear and quadratic contrasts and the treatments had 6 independent replicates (n = 6). Cell proliferation was significantly decreased by both SB2.5 and SB5 after 48h (−55% on average, P = 0.001) and 72h (−63% on average, P = 0.001). To assess the impact of SB on spontaneous differentiation, MSC were cultured for 27 d, with complete media change every 3 d. At 3 d, SB treated cells showed changes in morphology compared with controls, from spindle-shaped fibroblast-like to larger sail-shaped cells with intracellular shiny droplets. At 27d, cells were stained for osteocytic (Alizarin Red), chondrocytic (Alcian Blue) and adipocytic (Oil-Red-O) differentiation. No terminal differentiation was detected in MSC cultured in the BM alone, while accumulated lipids were clearly stained by Oil-Red-O in MSC cultured in the presence of SB. The phenotypic changes observed from 3 to 27 d were supported by a change in the pattern of gene expression, analyzed by semiquantitative real-time PCR. At 3 d, both SB2.5 and SB5 increased mRNA expression of peroxisome proliferator-activated receptor gamma (PPARg; P = 0.05, linear effect) and decreased osteocalcin (OC; P = 0.02, quadratic) and aggrecan (P = 0.04, quadratic) mRNA levels compared with untreated cells. At 27d, there was an increase in PPARg mRNA level (P = 0.02, linear) and decreased OC and aggrecan (P = 0.02 and P = 0.002, respectively, both linear and quadratic) with SB treatment. To conclude, our data suggest that SB promotes the differentiation of porcine bone marrow-derived MSC toward an adipocytic lineage.