Excessive fluoride exposure has been shown to induce diminished sperm quality and mitochondrial dysfunction. The interaction between mitochondria and the endoplasmic reticulum (ER) is critical for regulating mitochondrial function in spermatogenic cells. Therefore, this study was designed to investigate the molecular events involved in mitochondria-associated ER membranes (MAMs) in mice exposed to 25, 50, and 100 mg/L NaF for 60 days, and in GC-2spd treated with 1.5, 2.0, and 2.5 mM NaF for 24 hours. Mitochondrial stress tests revealed a significant reduction in basal respiration, maximal respiration, and ATP production, suggesting mitochondrial dysfunction following fluoride exposure. Results further indicated that fluoride exposure significantly enhanced ER-mitochondria contacts, mitochondrial Ca