Human umbilical cord perivascular cells (HUCPVCs) can differentiate along numerous lineages making them a favourable cell source for tissue regeneration. However, how these cells respond to biomechanical forces is unclear. This study aimed to determine whether cyclic stretch could regulate adipogenic differentiation of HUCPVCs, and to elucidate the mechanism of this regulation. In adipogenic culture, HUCPVCs expressed the adipocyte-specific transcription factors PPARgamma and C/EBPalpha and accumulated cytoplasmic lipid droplets. Exposure of these cells to equibiaxial cyclic stretch (10%, 0.5 Hz) in the presence of adipogenic medium, increased Smad2 phosphorylation compared to static samples and inhibited the expression of adipocyte markers; ERK1/2 phosphorylation was not changed. Inhibiting TGFbeta1 signaling decreased Smad2 phosphorylation and prevented the inhibition of adipogenic differentiation by cyclic stretch. These results demonstrate that cyclic equibiaxial stretch regulates HUCPVC differentiation even in the presence of an adipogenic milieu and should be an important consideration in developing future progenitor cell therapies.