Osteoarthritis (OA) is a degenerative disease characterized by deterioration of articular cartilage. Recent studies have demonstrated the importance of some microRNAs in cartilage damage. The aim of this study was to identify and characterize the expression of microRNA-634 (miR-634) in normal and OA chondrocytes, and to determine its role in OA pathogenesis. Human normal and OA chondrocytes obtained from patients were cultured in vitro. Transfection with miR-634 mimic or inhibitor was employed to investigate the effect of miR-634 on chondrocyte survival and matrix synthesis, and to identify miR-634 target. The results indicated that miR-634 was expressed at lower level in high grade OA chondrocyte compared with normal chondrocytes. Overexpression of miR-634 could inhibit cell survival and matrix synthesis in high grade OA chondrocytes. Furthermore, miR-634 targeted PIK3R1 gene that encodes the regulatory subunit 1 of class I PI3K (p85α) and exerted its inhibitory effect on the phosphorylation of Akt, mTOR, and S6 signal molecules in high grade OA chondrocytes. Therefore, the data suggested that miR-634 could suppress survival and matrix synthesis of high grade OA chondrocytes through targeting PIK3R1 gene to modulate the PI3K/Akt/S6 and PI3K/Akt/mTOR/S6 axes, with important implication for validating miR-634 as a potential target for OA therapy.