As the initial part in the development of osteoarthritis (OA), subchondral bone sclerosis has been considered to be initiated by excess mechanical loading and proven to be correlated to other pathological changes. Sclerostin, which is an essential mechanical stress response protein, is encoded by the SOST gene. It is expressed in osteocytes and mature chondrocytes and has been proven to be closely correlated to OA. However, the relationship and mechanism between the SOST gene and the development of OA remain unclear. The aim of the present study was to investigate the role of the SOST gene in OA pathogenesis in the subchondral bone. A knee anterior cruciate ligament transection (ACLT) mouse osteoarthritis (OA) model on SOST-knockout (SOST KO) and wild-type (WT) mice was established. The pathogenic and phenotypic changes in the subchondral bone were investigated by histology, micro-CT, immunohistochemistry, TRAP staining, Masson staining, and Toluidine blue staining. It was found that sclerostin expression decreased in both the calcified cartilage and mineralized subchondral structures during the development of OA. Joint instability induced a severe cartilage degradation phenotype, with higher OARSI scores in SOST KO mice, when compared to WT mice. SOST KO mice with OA exhibited a higher BMD and BV/TV ratio, as well as a higher rate of bone remodeling and TRAP-positive cell number, when compared to the WT counterparts, but the difference was not significant between the sham-operation groups. It was concluded that loss of sclerostin aggravates knee OA in mice by promoting subchondral bone sclerosis and increasing catabolic activity of cartilage.