Abstract: Objective To study the effect of C-type natriuretic peptide gene transfection on the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) so as to lay a foundation for finding a new method of MSC-based cartilage tissue engineering construction with high quality. Methods Bone marrow mesenchymal stem cells were isolated from Sprague-Dawley rats by whole bone marrow adherence method and were subcultured. Multi-differentiation capacity of the cells was identified by traditional osteogenic and adipogenic differentiation in vitro. The BMSCs were then transfected by recombinant adenovirus vector containing C-type natriuretic peptide gene and enhanced green fluorescent protein (Ad-NPPC-eGFP) with the specific multiplicity of infection (MOI). Flow cytometry was used to detect the transfection efficiency, real-time PCR was conducted to detect CNP mRNA expression, MTT assay was performed to examine the effect of transfection on cell proliferation,and alcian blue staining was performed to detect the extracellular matrix accumulation of the cells after chondrogenic differentiation in vitro for 14 days and 21 days. Results The rat BMSCs were successfully obtained, and osteogenic and adipogenic differentiation results revealed a good multi-differentiation capacity. Adenovirus transfected cells were in good condition with high transfection efficiency and no significant difference was shown on cell proliferation (P> 0.05). The expression of CNP mRNA in BMSCs after transfection was significantly higher in Ad-NPPC- eGFP- transfected group (P< 0.05). Alcian blue staining indicated significantly improved extracelluar matrix secretion (P< 0.05). Conclusion Recombinant adenovirus containing CNP and eGFP gene can be stably expressed in BMSCs with better chondrogenic differentiation ability in vitro.