Nephrolithiasis is a highly prevalent urological disease and results in a correspondingly heavy socioeconomic and healthcare burden. Calcium oxalate (CaOx) stones are among the most common types of kidney stones. They are associated with renal tubular damage, interstitial fibrosis and chronic kidney disease (CKD). However, the molecular mechanisms in CaOx crystal deposition-induced renal fibrosis remain unclear. Chemokines and their receptors act a crucial role in the progression of renal fibrosis through inflammatory cell infiltration, autophagy activation, and epithelial-mesenchymal transition (EMT). The current work aims to study the action and mechanism of the C-X-C motif chemokine receptor 4 (CXCR4) in CaOx crystal deposition-induced renal fibrosis. Transcriptome RNA sequencing, qPCR, and immunohistochemistry revealed that the expression of CXCR4 was significantly upregulated in patients with nephrolithiasis and hyperoxaluric mice. Renal injury and fibrosis were significantly suppressed by inhibiting CXCR4 with AMD3100 or siRNA in hyperoxaluric mice and oxalate-stimulated HK-2 cells; EMT, reactive oxygen species (ROS) levels, and autophagy were also suppressed. Bioinformatic analysis revealed that the NF-κB pathway was activated in hyperoxaluric mice. Mechanistically, activation of the NF-κB pathway was suppressed by CXCR4 inhibition in CaOx crystal-induced renal fibrosis; this suppression was significantly aggravated by the NF-κB inhibitor BAY-11-7085. Moreover, inhibition of autophagy attenuated EMT progression in vitro. Our results suggest that CXCR4 inhibition attenuates CaOx crystal deposition-induced renal fibrosis by suppressing autophagy and EMT through the NF-κB pathway. Therefore, CXCR4 is a potential target for preventing renal fibrosis in patients with nephrolithiasis.