Osteoporosis is a systemic bone disease characterized by decreased bone mass and deterioration of bone microstructure, which leads to increased bone fragility and increased risk of fractures. Casein kinase 2 interacting protein 1 (CKIP-1, also known as PLEKHO1) is involved in the biological process of bone formation, differentiation and apoptosis, and is a negative regulator of bone formation. QiangGuYin (QGY) is a famous TCM formula that has been widely used in China for the clinical treatment of postmenopausal osteoporosis for decades, but the effect in regulating CKIP-1 on osteoporosis is not fully understood. This study aimed to explore the potential mechanism of CKIP-1 participating in autophagy in bone cells through the AKT/mTOR signaling pathway and the regulatory effect of QGY. The results in vivo showed that QGY treatment can significantly improve the bone quality of osteoporotic rats, down-regulate the expression of CKIP-1, LC3II/I and RANKL, and up-regulated the expression of p62, p-AKT/AKT, p-mTOR/mTOR, RUNX2 and OPG. It is worth noting that the results in vitro confirmed that CKIP-1 interacts with AKT. By up-regulating the expression of Atg5 and down-regulating the p62, the level of LC3 (autophagosome) is increased, and the cells osteogenesis and differentiation are inhibited. QGY inhibits the combination of CKIP-1 and AKT in osteoblasts, activates the AKT/mTOR signaling pathway, inhibits autophagy, and promotes cell differentiation, thereby exerting an anti-osteoporosis effect. Therefore, QGY targeting CKIP-1 to regulate the AKT/mTOR-autophagy signaling pathway may represent a promising drug candidate for the treatment of osteoporosis.