It has been discussed whether reduced glutathione (GSH) could promote the chondrogenic differentiation ability of human umbilical cord mesenchymal stem cells (hUC-MSCs). hUC-MSCs were isolated from human umbilical cord and their specificity was identified, then induced into cartilage-like cells in chondrogenic induction medium with transforming growth factor beta 1 (TGF-β1), especially with GSH. The morphological change before and after induction was observed through inverted phase contrast microscope, Type II collagen (COL2-A1) and glycosaminoglycan (GAG) were analyzed qualitatively by Toluidine blue and immunofluorescence technique, respectively, the contents of COL2-A1 and GAG were estimated from the determination of hydroxyproline content and Alcian Blue method separately. The mRNA expressions of GAG and COL2-A1 were assayed by real-time fluorescence quantitative PCR. After continuously cultured for 21 days with GSH, Toluidine blue staining and immunofluorescence reaction were all positive in basic induction medium group (group B), basic induction medium +0.5% dimethylsulfoxide (DMSO) group (group BD) and basic induction medium +0.5% DMSO +500 μM GSH group (group BDG). Moreover, compared with group B and group BD, the contents of COL2-A1 and GAG in group BDG relatively increased and the mRNA expression level of COL2-A1 and GAG also comparatively increased (P < 0.05) and both had a significant statistical significance (P < 0.05). So GSH might promote the induction of hUC-MSCs to differentiate into cartilage-like cells.