Objective To determine the effect of Janus activated tyrosine kinase 2 and signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway on the metabolism and mitochondrial oxidative stress in the chondrocytes of osteoarthritis (OA) mice in order to explore the role of the signaling pathway in this process. Methods Ten C57BL/6 mice were randomly and equally divided into OA model group and normal control group. In 3 weeks after the establishment of OA model, all mice were sacrificed and their knee joint synovium tissues were harvested to isolate chondrocytes. SC-39100, a JAK2/STAT3 Signaling pathway agonist, was used to treat the obtained chondrocytes from the both groups of mice. The expression of p-JAK2, p-STAT3, Bcl-2, Bax, succinate dehydrogenase (SDH), and cytochrome c oxidase (COX) was detected by Western blotting. The content and activity of malondialdehyde (MDA) were measured. Results Compared with the chondrocytes derived from control group, the OA chondrocytes had significantly lower protein levels of p-JAK2, p-STAT3 and Bcl-2, higher level of Bax, lower SDH and COX, and increased content of MDA (all P＜0.05). The treatment of SC-39100 enhanced the expression of p-JAK2, p-STAT3 and Bcl-2, decreased the expression of Bax, up-regulated the levels of SDH and COX, and reduced the content of MDA (all P＜0.05). However, the treatment had no effect on all indices in the chondrocytes from control group (all P＞0.05). Significant differences were found in the above all indices between the OA chondrocytes after SC-39100 treatment and the chondrocytes from control group (P＜0.05). Conclusion The JAK2/STAT3 signaling pathway is closely associated with OA chondrocytes. Its activation can suppress apoptosis in chondrocytes and enhance the anti-oxidative stress capacity of chondrocyte mitochondria.