Titanium dioxide nanoparticles (TiO2-NPs) are widely utilized as a whitish pigment with potential applications in various sectors, including cosmetics, pharmaceuticals, food production, textiles, and water treatment. They are commonly employed as a sanitizing agent for microorganism control. Due to their small size and expansive surface area, these nanoparticles can inflict severe toxic effects on various human organs and cause serious damage to the surrounding environment. This study investigated the gonadal toxicity induced by TiO2-NPs in male Sprague Dawley rats. Accordingly, TiO2-NPs were employed with an average particle size of 55 nm. Twenty-five adult male Sprague Dawley rats weighing 107 ± 120 g underwent a 7-day acclimatization period and were subsequently randomly divided into five groups: The Control group (C), which received no treatment; the saline-treated group (S), treated with normal saline; and three treatment groups (G1, G2, and G3), each consisting of five rats. The rats were exposed to different concentrations of TiO2-NPs for 28 days on alternate days, determined after establishing the LC50 value. TiO2 was administered intraperitoneally with injection at doses of 50, 90, and 130 mg/kg body weight. The blood samples were collected at the beginning and end of the experiment. The 28-day exposure results revealed severe histological damage in testicular tissues at high and medium doses compared to the control and saline-treated groups. These damages included widened lumen, basement membrane damage, necrosis, and vacuolation in the tissues, which were highly significant in both the G1 and G2 groups. This study demonstrates the toxic effects of titanium dioxide nanoparticles on the gonads of Sprague Dawley rats. It is concluded that the selected concentrations of TiO2 negatively impact the health of living organisms.