Background: Renal calculi, a painful kidney stone disease has worldwide health hazard. Cucurbita pepo is used for the management of lower urinary tract disease, benign prostatic hyperplasia, and micturition difficulty. Objective: The present study was investigated to resolve the antiurolithic potential of methanol extract of C. pepo (MECP) seed against sodium oxalate-induced renal calculi using both in vitro and in vivo models. Materials and Methods: MECP was prepared by macerating C. pepo seed powder in methanol. Gas chromatography coupled with mass spectrometry (GC-MS) analysis was performed to characterize the phytochemical profile of MECP. In vitro techniques such as nucleation, aggregation, growth, and dissolution assays were performed using different concentrations (20, 40, 60, and 80 mg/mL) of MECP and the standard drug cystone to determine their calcium oxalate (CaOx) crystals inhibitory potential. In male albino rats, calculi were induced by intraperitoneal administration of sodium oxalate (70 mg/kg) for 10 days. Various doses (250, 500, and 1000 mg/kg) of MECP were administered orally to male albino rats. Various pathological parameters such as body and kidney weights, serum (creatinine, blood urea nitrogen, and uric acid), urinary (calcium, potassium, oxalate, sodium, magnesium, phosphate, pH, and volume) analysis, and kidney histopathology were executed. Results: GC-MS fingerprints showed that beta-tocopherol, stigmasterol, and squalene are the major phytochemicals found in MECP. Results demonstrated that MECP significantly inhibited various steps of CaOx crystal formation such as nucleation, aggregation, growth, and dissolution in dose-dependent manner. MECP normalized the raised levels of oxalate, calcium, sodium, phosphate, uric acid, restored alterations in histopathology while elevated the reduced levels of magnesium, urine volume, and pH. Conclusion: The undertaken study rationalized the usage of C. pepo as an alternative or adjuvant treatment for renal calculi after clinical trials in human subjects.