Mechanical overloading and aging are two essential factors for osteoarthritis (OA) development. Mitochondria has been identified as a mechano-transducer situated between extracellular mechanical signals and chondrocyte biology, but its role and mechanism in mechanical stress-associated chondrocyte senescence and OA is not elucidated. Herein, we found that PDZK1, one of the PDZ proteins which belongs to NHE Regulatory Factor (NHERF) family, is a key factor in biomechanically induced mitochondria dysfunction and chondrocyte senescence during OA progression. PDZK1 is reduced by mechanical overload, and is decreased in articular cartilage of OA patients, aged mice, and OA mice. Knockout of Pdzk1 in chondrocytes aggravates mechanical overload-induced cartilage degeneration, whereas intraarticular injection of adeno-associated virus-expressing Pdzk1 had a therapeutic effect. Moreover, we found loss of PDZK1 impaired chondrocytes mitochondria function with accumulated damaged mitochondria, decreased mtDNA content and increased production of reactive oxygen species. Supplementary of PDZK1 or mitoQ alleviated chondrocyte senescence and cartilage degeneration and significantly protected chondrocyte mitochondrial functions. MRNA sequencing in articular cartilage from Pdzk1KO mice and controls showed that PDZK1 deficiency in chondrocytes interference with mitochondrial function through binding and inhibiting Hmgcs2. Our results suggest that PDZK1 deficiency plays a crucial role in mediating excessive mechanical load induced chondrocyte senescence and is associated with mitochondrial dysfunction. Overexpression of PDZK1 or preservation mitochondrial functions by MitoQ might present a new therapeutic method for mechanical overload induced OA.