Calcium is an essential regulating factor in a variety of biological functions including reproduction. It is widely required for different physiological activities in spermatozoa including spermatogenesis, sperm motility, capacitation, acrosome response, and fertilization. Recent advancements in nanotechnology have broadened its potential applications in biomedicine, including improving animal reproductive aspects. Our research was planned fundamentally to examine the influence of calcium deficiency and calcium administration using Nano and commercial calcium in two doses for each type of calcium (1000 mg/Kg body weight (BW) and 500 mg/Kg BW once daily for 64 days orally) to reveal their effects on male reproductive function as well as regulatory mechanisms connected to male fertility. Semen examination, biochemical analyses, enzymatic antioxidant, lipid peroxidation, and testis histopathology were all evaluated after 64 days. Our results revealed that Serum calcium, testosterone, and ABP levels, sperm count, motility, and percentage of intact acrosomes, as well as testicular antioxidant enzymes, were all considerably lower in the calcium-free diet group, whereas sperm abnormalities and testicular Malondialdehyde were significantly higher. In calcium (Nano and commercial) administered male rats, serum calcium, testosterone, and ABP levels, sperm count, motility, percentage of intact acrosomes, and testicular antioxidant enzymes all increased significantly, whereas sperm abnormalities and testicular Malondialdehyde dropped dramatically. Overall, these results point to a strong link between Ca²⁺, sperm function, and fertility outcomes. what's more, Ca²⁺ supplementation, particularly nanoparticles could efficiently improve male reproductive function and fertility.