Osteogenesis of renal tubular epithelial cells (RTEC) is an important trigger for calcium oxalate (CaOx) kidney stone formation, but whether macrophages are involved in RTEC osteogenesis is unclear. The purpose of this study was to investigate the role and mechanism of macrophages in CaOx kidney stones on RTEC osteogenesis. Oxalate or ethylene glycol was used to construct in vitro and in vivo CaOx kidney stone models, respectively. Macrophage-derived conditioned medium was used to induce osteogenesis in HK-2 cells, and genetic controls and pharmacological interventions were used to investigate the underlying mechanism. The results demonstrated that macrophage-conditioned medium under oxalate intervention facilitated the increase of alkaline phosphatase and calcium salts as well as the upregulation of osteogenic marker genes (BMP2 and RUNX2) expression in HK-2 cells. On the one hand, the knockdown of the JAK2 gene in HK-2 cells reverses the role of macrophage-derived conditioned medium in promoting osteogenesis in HK-2 cells. On the other hand, inhibition of prostaglandin E2 (PGE2) generation in macrophages reverses osteogenesis in HK-2 cells. Moreover, inhibition of PGE2 generation would cure ethylene glycol-induced renal injury and calcium salt deposition, as well as osteogenesis of RTEC. This study illustrates that in the presence of oxalate, macrophages secret PGE2 to activate JAK2/STAT3 signaling in RTEC, which could trigger osteogenesis. It provides new insights into the mechanism of CaOx kidney stone formation.