Purpose Capsaicin (CAP) is the primary pungent alkaloid found in chili peppers. The aim of this study is to screen for candidate therapeutic agents targeting the mitochondrial dysfunction–pyroptosis axis in diabetic nephropathy (DN) and to validate the efficacy and mechanism of action of capsaicin. Methods Bioinformatics analysis was conducted to identify candidate genes and drugs. The protective effects and underlying mechanisms of CAP were investigated using a high-glucose (HG)-induced HK-2 cell model and a streptozotocin-induced T1DM DN rat model. Experimental techniques included CCK8 assay, flow cytometry (pyroptosis and ROS), JC-1 staining, Western blot analysis, and histopathological staining. GPX4-IN-3 was applied in vivo and in vitro to determine whether it reversed the protective effects of capsaicin. The data were analyzed using the Shapiro–Wilk test, one-way analysis of variance (ANOVA) with corresponding post-hoc tests, and correlation analysis. Results Bioinformatic analysis identified 12 intersecting genes and highlighted CAP as a potential GPX4-targeting compound. Molecular docking demonstrated a high binding affinity of CAP for GPX4 and caspase-9. In vitro , CAP dose-dependently restored cell viability and suppressed HG-induced pyroptosis in HK-2 cells. It significantly alleviated mitochondrial damage by reducing ROS production, restoring mitochondrial membrane potential, and improving mitochondrial ultrastructure. Notably, these protective effects were reversed by GPX4-IN-3. Western blot analysis revealed that CAP upregulated GPX4 expression and inhibited activation of the NLRP3/caspase-1/GSDMD-N pyroptosis pathway. In vivo , CAP treatment markedly improved renal histopathology and renal function in DN rats; however, these effects were attenuated by co-administration of GPX4-IN-3. Conclusion This study demonstrates that CAP ameliorates diabetic renal injury by targeting GPX4, thereby mitigating mitochondrial dysfunction and subsequent NLRP3-mediated pyroptosis. These findings validate the bioinformatic prediction and reveal a novel mechanism of action for CAP, positioning it as a promising therapeutic candidate for DN.