Trichostatin A (TSA) is a histone deacetylase inhibitor (HDACi) known to modulate differentiation of many cells. However, its effect on chondrogenesis remains elusive. This study was aimed to investigate the effects of TSA on in vitro transforming growth factor-β1 (TGF-β1)-induced chondrogenesis of human mesenchymal stem cells (hMSCs). The pellet cultures of hMSCs in a chondrogenic medium were exposed to TGF-β1 and TSA. Quantitative reverse transcription/polymerase chain reaction (PCR) analysis, Alcian blue staining, and immunohistochemistry staining were used to confirm and compare the differences in chondrogenesis by analyzing the mRNA of chondrogenic genes (Sox9, Aggrecan, and Col2A1), synthesis of chondrogenic proteins and type II collagen, respectively. TGF-β1 signaling and its downstream targets were determined by western blot analysis. TGF-β1 led to significant increases in chondrogenic gene expression and the synthesis of chondrogenic proteins. However, TSA significantly decreased chondrogenic gene expression and the synthesis of chondrogenic proteins in a dose-dependent manner. TGF-β1 increased phosphorylation of Smad 2/3 and Sp1 expression around half an hour after induction. The increase of Sp1, but not Smad 2/3 activation was almost completely blocked by the addition of TSA. The chondrogenic effect of TGF-β1 was also suppressed by the Sp1-binding inhibitor mithramycin A. Finally, overexpression of Sp1 abolished TSA-mediated inhibition of TGF-β1-induced chondrogenesis. Our study showed that TSA inhibited chondrogenesis through inhibition of TGF-β1-induced Sp1 expression. Furthermore, Sp1 could be a useful tool in future studies looking into biological mechanisms by which chondrogenesis of hMSCs can be augmented, especially in the area of clinical application.