Background aims. Human (h) adipose tissue-derived mesenchymal stromal cells (ASC) constitute an interesting cellular source for bone tissue engineering applications. Wnts, for example Wnt5a, are probably important regulators of osteogenic differentiation of stem cells, but the role of Wnt5a in hASC lineage commitment and the mechanisms activated upon Wnt5a binding are unknown. We examined whether Wnt5a induces osteogenic and/or adipogenic differentiation of hASC. Methods. hASC were incubated for 7 days with or without Wnt5a, rho-associated kinase (ROCK)-activity inhibitor Y27632 or Wnt3a. Cells were lysed for total RNA isolation, DNA content and alkaline phosphatase (ALP) activity. Mineralized nodule formation and gene expression of osteogenic markers osteocalcin and runt-related protein-2 (RUNX2), and adipogenic markers peroxisome proliferator activator receptor-γ (PPARγ) and transcription factor apetala-2 (aP2), were analyzed. hASC were incubated with Wnt5a or Wnt3a to determine activation of canonical and/or non-canonical Wnt signaling pathways, and protein kinase C activity (PKC), total β-catenin content and gene expression of connexin 43 and cyclin D1 were quantified. Results. Wnt5a increased ALP activity and RUNX2 and osteocalcin gene expression, and down-regulated adipogenic markers through ROCK activity. Wnt5a also induced mineralized nodule formation. Wnt3a only enhanced RUNX2 and osteocalcin gene expression, and did not induce osteogenic differentiation. Wnt5a activated the non-canonical Wnt signaling pathway by increasing PKC activity, while Wnt3a mildly activated the Wnt canonical pathway by increasing total β-catenin content and connexin 43 and cyclin D1 gene expression. Conclusions. Our data illustrate the importance of Wnt5a as a stimulator of hASC osteogenic differentiation, and show that changes in actin cytoskeleton controlled by ROCK are determinants for Wnt5a-induced osteogenic differentiation of hASC.