This study aims to investigate the protective effects and mechanisms of icariin against high-fat diet (HFD)-induced impairment of Sertoli cell tight junction in mice. Seven-week-old male C57BL/6J mice were randomly divided into a normal diet group, high-fat diet model group, and HFD groups supplemented with low-does group (3 mg/kg), medium-does group (9 mg/kg), and high-doses group (27 mg/kg) of icariin. Organ indices of the testes, epididymis, and seminal vesicles were calculated, and sperm concentration and motility were analyzed. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (γ-GT), and glucose (GLU) in serum were measured by ELISA. Testicular morphology was examined by H&E staining, and the expression levels of tight junction proteins zonula occludens-1 (ZO-1), Occludin, and Claudin-11 were detected by Western blot. In vitro, a tight junction injury model was established in mouse Sertoli cells (TM4) using palmitic acid. A CCK-8 assay was used to screen the optimal intervention doses of icaritin (0.2, 0.4, 0.8 μmol/L). Western blot was used to measure the expression levels of tight junction proteins, interleukin-1β (IL-1β), IL-18, and key proteins in the NF-κB/NLRP3 pathway in both mouse testes and TM4 cells. Transepithelial electrical resistance and fluorescein isothiocyanate-dextran flux were assessed to monitor cell permeability, while RT-qPCR detected mRNA levels of the inflammatory factors TNF-α, IL-6, and IL-33. In vivo results showed that icariin significantly improved the epididymal index, sperm concentration and motility, ameliorated testicular histopathology and serum biochemical abnormalities, and up-regulated the expression of tight junction proteins. In vitro experiments demonstrated that icariin upregulates the expression of tight junction-related proteins in TM4 cells, thereby improving cellular barrier integrity by reducing cell permeability. Concurrently, icaritin suppressed the mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-33), as well as the protein expression of key components of the NF-κB/NLRP3 signaling pathway. In conclusion, icariin alleviates HFD-induced damage to Sertoli cell tight junctions, potentially by mitigating the NF-κB/NLRP3 pathway-mediated inflammatory response.