Aims: We aimed at exploring the role of nicotinamide adenine dinucleotide phosphate oxidase subunit 4 (Nox4) in the regulation of hypercalciuria-induced renal oxidative damage and crystal depositions.Results: High calcium activated Nox4 expression through protein kinase C (PKC). Downregulation of Nox4 expression attenuated hypercalciuria-induced osteoblast-associated protein expression, oxidative stress injury, and crystal deposition in rat kidneys of 1,25-dihydroxyvitamin D3 (VitD) urolithiasis model. Further, calcium-induced activation of mitogen-activated protein kinase (MAPK), overexpression of osteoblast-associated protein, oxidative stress injury, apoptosis, and calcium salt deposition in normal rat kidney epithelial-like (NRK-52E) cells were reversed by downregulating Nox4 expression but were enhanced by upregulating Nox4 expression in vitro. Moreover, calcium-induced increases of osteoblast-associated protein expression were attenuated by the c-Jun-N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) inhibitors.Innovation: Our results demonstrated the effect of Nox4 in the pathological process of kidney stones in in vitro and in vivo studies for the first time. Calcium aggravates renal oxidative stress injury and crystal deposition by activating the Nox4-related reactive oxygen species (ROS)-ERK/JNK pathway in the rat kidney. This study is expected to provide a new theoretical basis for the prevention and treatment of kidney stones.Conclusion: Nox4-derived ROS induced by calcium through PKC caused oxidative stress damage and apoptosis in renal tubular epithelial cells; in addition, Nox4-derived ROS induced by calcium mediated abnormal activation of the bone morphogenetic protein 2 (BMP2) signaling pathway through the MAPK signaling pathway, which induced renal tubular epithelial cells to transdifferentiate into osteoblast-like cells, resulting in the formation of a kidney stone.