Oxidative stress is considered to be an important cause of dysfunction in chondrocytes and articular cartilage degradation, which leads to the pathogenesis of osteoarthritis (OA) and cartilage aging. The present study aimed to assess the effects of the widely applied antioxidant, ascorbic acid (AA), on human chondrocytes against hydrogen peroxide (H2O2) in vitro. Using annexin V‑fluorescein isothiocyanate, 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyl tetrazolium bromide and senescence‑associated β‑galactosidase assays, the present study identified that AA reduced apoptosis, reduced the loss of viability and markedly decreased H2O2‑mediated senescence in cells treated with H2O2. Furthermore, AA not only stimulated the expression levels of collagens and proteoglycans, but also inhibited the differentiation of chondrocytes under conditions of oxidative stress. In addition, reverse transcription‑quantitative polymerase chain reaction and western blotting demonstrated that AA decreased the activity of nrf2, NF‑κB, AP1 and matrix metalloproteinase‑3, which is stimulated by H2O2. In conclusion, AA efficiently protected human chondrocytes against damage induced by H2O2 by regulating multiple regulatory pathways.