The Wnt/β-catenin signaling pathway plays critical roles in self-renewal and differentiation of mesenchymal stem cells. However, very little is known about its role in the chondrogenesis of human adipose-derived stem cells (hADSCs). In this study, we analyzed protein expression of several key components of the Wnt/β-catenin signaling pathway using a 21-day in vitro model of hADSC chondrogenesis. Wnt1, β-catenin, and GSK3β levels increased sharply at day 12, peaked at day 18, and then declined. Expression of TCF1, a target gene of Wnt/β-catenin signaling, closely followed that of Wnt1. These results were consistent with changes in endonuclear β-catenin levels. Gene expression of the chondrocyte-specific markers, collagen type II (COL II), SOX9, and aggrecan, increased during hADSC chondrogenesis, peaked at day 12, and then declined. Adding a Wnt inhibitor (days 0–21) resulted in consistently elevated levels of COL II, SOX9, and aggrecan mRNA. In contrast, adding Wnt1 (days 0–21) to cultures led to sustained Wnt/β-catenin signaling over the 21 days and suppressed expression of chondrocyte-specific markers. Moreover, adding Wnt1 at late stages of differentiation (day 18) further diminished chondrocyte-specific marker expression. Together, these results showed that inactivation of Wnt/β-catenin signaling is needed for the progression of chondrogenesis and the maturation and phenotype maintenance of chondroid cells.