Mesenchymal stem cells (MSCs) are the common precursors of several functionally disparate cell lineages. A plethora of chemical and physical stimuli contribute to lineage decisions and guidance, including mechanical stretch concomitant with physical movement. Here, we examined how stretch regulates MSC differentiation into adipocytes and the intracellular signaling pathways involved. MSCs were cultured under adipogenic conditions and divided into a control and an experimental group. Cultures in the experimental group were subjected to a sinusoidal stretch regimen delivered via flexible culture bottoms (5% magnitude, 10 times per min, 6 h/day, 3 or 5 days). Expression levels of the adipocyte markers PPARγ-2, adiponectin, and C/EBPα were measured as indices of differentiation. Compared to controls, MSCs exposed to mechanical stretch exhibited downregulated PPARγ-2, adiponectin, and C/EBPα mRNA expression. Alternatively, stretch upregulated phosphorylation of Smad2. This stretch-induced increase in Smad2 phosphorylation was suppressed by pretreatment with the TGFβ1/Smad2 pathway antagonist SB-431542. Pretreatment with the TGFβ1/Smad2 signaling agonist TGFβ1 facilitated the inhibitory effect of stretch on the expression levels of PPARγ-2, adiponectin, and C/EBPα proteins, while pretreatment with SB-431542 reversed the inhibitory effects of subsequent stretch on the expression levels of these markers. These results strongly suggest that the anti-adipogenic effects of mechanical stretch on MSCs are mediated, at least in part, by activation of the TGFβ1/Smad2 signaling pathway.