Nano-antimony trioxide (Nano-Sb₂O₃) is extensively utilized in industrial production and consumer products, leading to widespread environmental contamination and human exposure. Accumulating evidence has demonstrated that Nano-Sb₂O₃ induces male reproductive toxicity, yet the underlying molecular mechanisms remain poorly understood. In this study, male C57BL/6 mice were exposed to Nano-Sb₂O₃ (2.5, 5.0, and 7.5 mg/kg/day). Exposure to Nano-Sb₂O₃ induced dose-dependent reproductive toxicity, evidenced by dose-dependent reductions in sperm motility (56.70% to 36.10%) and sperm density (15.76 × 10⁶/mL to 2.79 × 10⁶/mL) and a marked elevation in sperm malformation rates (4.56% to 44.36%), as well as severe histopathological alterations, testicular Sb accumulation, and elevated reactive oxygen species (ROS) levels. Transcriptomic analysis revealed significant enrichment of the PPAR and PI3K-Akt signaling pathways and identified SPP1 as one of the most significantly differentially expressed genes. Computational pathway perturbation analyses further yielded hypothesis-generating evidence supporting the potential involvement of PPAR signaling suppression and PI3K-Akt and inflammatory pathway activation following Nano-Sb₂O₃ exposure. Both mRNA and protein levels of SPP1 were significantly upregulated in a dose-dependent manner in mouse testes and TM4 Sertoli cells. In vitro experiments further demonstrated that Nano-Sb₂O₃ increased the expression of pro-inflammatory cytokines IL-1β and IL-6 by up to 5.6-fold and 4.7-fold, respectively, while impairing Sertoli cell viability and wound-healing capacity. Importantly, Spp1 silencing attenuated inflammatory responses and restored the expression of blood–testis barrier (BTB)-associated proteins, including ZO-1, Claudin-11, and N-cadherin. These findings suggest that SPP1 may contribute to Nano-Sb₂O₃-induced inflammatory responses and alterations in BTB-associated proteins, thereby potentially participating in male reproductive injury.