Atractylenolide-III (AT-III) is well known as its role in antioxidant and anti-inflammatory. Present study was aimed to figure out its effects on osteoarthritis and potential mechanisms. Rat model, human osteoarthritis cartilage explants as well as rat/human chondrocyte cultures were prepared to test AT-III's effects on osteoarthritis progression and chondrocyte senescence. Potential targeted molecules of AT-III were predicted using network pharmacology and molecular docking, assessed by Western blotting and then verified with rescue experiments. AT-III treatment alleviated osteoarthritis severity (shown by OARSI grading score and micro-CT) and chondrocyte senescence (indexed by levels of SA-beta-gal, P16, P53, MMP13, ROS and ratio of healthy/collapsed mitochondrial membrane potentials). Network pharmacology and molecular docking suggested that AT-III might play role through NF-kappa B pathway. Further experiments revealed that AT-III reduced phosphorylation of IKK alpha/beta, I kappa B alpha and P65 in NF-kappa B pathway. As well as nuclear translocation of p65. Both in vivo and in vitro experiments indicated that AT-III's effects on osteoarthritis and anti-senescence were reversed by an NF-kappa B agonist. AT-III could alleviate osteoarthritis by inhibiting chondrocyte senescence through NF-kappa B pathway, which indicated that AT-III is a prospective drug for osteoarthritis treatment.