Multilineage-differentiating stress-enduring (Muse) cells are stage-specific embryonic antigen-3 (SSEA-3) positive cells existing in mesenchymal stem cell (MSC) populations. Muse cells have the pluripotency to differentiate into all germ layers as embryonic stem cells. In this study, we aimed to investigate the efficacy of Muse cell transplantation for osteochondral defect repair. Muse cells were isolated from human bone marrow MSCs. An osteochondral defect was created in the patellar groove of immunodeficient rats. After this, cell injection was performed, whereby rats were divided into 3 groups: the control group, the rats of which were given a PBS injection; the non-Muse group, which comprised 5 × 104 SSEA-3 negative non-Muse cells; and the Muse group, which comprised 5 × 104 SSEA-3 positive Muse cells. The white repaired tissue had a mostly smooth homogenous surface at 12 weeks after treatment in the Muse group, while no repair tissue was detected in the control and non-Muse groups. Histological assessments showed better repair at the cartilage defect sites in the Muse group compared to the other groups at 4 and 12 weeks after treatment. Muse cells could be a new promising cell source for the treatment of osteochondral defects.