In the present study, we investigated the effect of low-frequency electroacupuncture (EA) on the differentiation of bone mesenchymal stem cells (BMSCs) into chondrocytes and the molecular mechanism involved. We isolated BMSCs from Sprague-Dawley (SD) rat bone marrow. Third-generation SD rat BMSCs (P3 BMSCs) were harvested and characterized by flow cytometry with FITC staining. Data indicated that the positive rates of CD90 and CD45 were 98.22 and 1.91%, respectively, indicating the high purity of the BMSCs. The P3 BMSCs were treated with EA for 15 or 30 min daily for 7 or 14 days. Using optical microscopy and transmission electron microscopy, we found that EA induced morphological changes in the BMSCs, displaying typical morphology of early chondrocytes. In addition, we found that the cytoplasm and extracellular matrices were metachromatically stained by toluidine blue in the treated cells in a dose-dependent manner, indicating that EA treatment resulted in the expression of glycosaminoglycan. Furthermore, upon immunohistocytochemical staining and Western blotting, we found that EA treatment significantly and dose-dependently induced expression of chondrocyte-specific matrix protein type II collagen, which may have been mediated by the transcription factor Sox9, as the mRNA expression of Sox9 was found to be significantly increased after EA treatment. Taken together, these results suggest that EA can be employed as a novel non-drug-inducing method for the differentiation of BMSCs into chondrocytes.