Osteoarthritis (OA) is a degenerative joint disease that usually occurs in the elderly, and docosahexaenoic acid (DHA) plays a therapeutic role in cardiovascular disease, diabetes, and rheumatoid arthritis (RA) with its anti-inflammatory and antioxidant effects. The objective of this study is to investigate the effect and mechanism of DHA on hypertrophic differentiation and senescence of OA chondrocytes to provide a theoretical basis for the effect of OA clinical treatment. A human OA chondrocyte model was established by IL-1 & beta;, and a rat model of OA was established by anterior cruciate ligament (ACL) transection and medial meniscectomy. The result showed DHA promoted chondrocyte proliferation and reduced apoptosis. Transmission electron microscopy (TEM) analysis showed that there were more autophagosomes in the cytoplasm under the treatment of DHA. Compared to the OA group, samples from the OA + DHA group showed thickened cartilage, reduced degeneration, and an increased rate of collagen II-positive cells, while the Mankin score was significantly lower. In addition, DHA decreased the expression of phosphorylated mammalian target of rapamycin (p-mTOR) and the ratio of light chain 3-I/II (LC3-I/II) and increased the expression of Beclin-1 and Bcl-2 measured by western blot analysis. Therefore, DHA promotes chondrocyte proliferation, reduces apoptosis, and increases autophagy in OA chondrocytes, a process that is accomplished by inhibiting the expression of mTOR, c-Jun N-terminal kinase (JNK), and p38 signaling pathways, providing new perspectives and bootstrap points for the prevention and treatment of OA.