<p>Excessive fluoride exposure is associated with male reproductive dysfunction, but the underlying mechanisms remain poorly understood. This study investigated the role of Inositol 1,4,5-trisphosphate receptor (IP3R)-mitochondrial calcium uniporter (MCU)-mediated mitochondrial calcium (Ca²⁺) overload in fluoride-triggered apoptosis in spermatogonia. The results demonstrated that fluoride induced oxidative stress, decreased mitochondrial membrane potential, and triggered apoptosis in spermatogonia. Furthermore, we observed that fluoride exposure induced calcium transfer from the endoplasmic reticulum (ER) to the mitochondria, leading to excessive mitochondrial Ca²⁺ accumulation. Mechanistic studies revealed that fluoride activated the IP3R-MCU signaling pathway, a key regulator of Ca²⁺ release from the ER. Notably, treatment with the calcium chelator BAPTA-AM significantly inhibited mitochondrial Ca²⁺ overload and protected spermatogonia against fluoride-induced apoptosis. In conclusion, the findings indicated that fluoride activated the IP3R-MCU pathway, promoted Ca²⁺ release from the ER, and led to mitochondrial calcium overload, inducing oxidative stress and apoptotic factor release, ultimately triggering apoptosis in spermatogonia. These findings offer potential therapeutic targets for mitigating fluoride-induced reproductive toxicity.</p> Graphical Abstract <p></p>