Abstract: Objective To determine the effects of exogenous nitric oxide (NO) donor sodium nitroprusside(SNP) on the proliferation and expression of related-gene of adipose-derived stem cells(ADSCs) and its role in chondrogenic differentiation of ADSCs. Methods Rat ADSCs were harvested and cultured, and then induced to osteogenic and adipogenic differentiations, detected with Alizarin red stained and Oil red O stain, respectively. The change of NO during chondrogenic differentiation of ADSCs was tested by NO detection kit. Cell counting kit-8 (CCK8) was used to detect the proliferation of ADSCs under different concentrations of SNP (0.25 mmol/L, 1.00 mmol/L, 4.00 mmol/L) . Gene expression level of transformation growth factor (TGF-β1) and specific gene of chondrogenic differentiation-signaling protein Smad3 and Collage Ⅱα1 (Col-Ⅱα1), were detected by Real time-PCR (RT-PCR) method. Results Positive alizarin red staining and Oil red O staining were found after osteogenic and adipogenic induction of cultured ADSCs. Higher concentrations of NO were found in the supernatant of the experimental group with ADSCs chondrogenic differentiations compared with the controls (P <0.05). Low concentrations (0.25 mmol/L,1.00 mmol/L) of SNP showed no significant effects on cell proliferations (P>0.05), whereas high concentration (4.00 mmol/L) of SNP inhibited cell proliferation (P <0.05). RT-PCR revealed that SNP inhibited the gene expression of TGF-β1mRNA and chondrogenic differentiation of specific geneSmad3mRNA, Col-Ⅱα1mRNA. Conclusion SNP can inhibit chondrogenic differentiations by suppressing the production of TGF-β1 and inhibiting downstream of TGF-β1 signaling pathways, thereby inhibiting ADSCs differentiation into chondrocytes.