AbstractNumerous effects of “titanium dioxide nanoparticles (TiO2 NPs)” have been described and significantly utilized in complementary drugs for many decades. This study is conducted to analyze the therapeutic efficiency of TiO2 NPs against the acetaminophen‐(AMP) induced toxicity. TiO2 NPs were initially prepared using Ginkgo biloba leaf extract as reducing and stabilizing agent. The prepared TiO2 NPs were studied using various spectroscopic and microscopic techniques. AFM, TEM, XRD, and DLS studies have confirmed the formation of TiO2 NPs with size between 20 to 60 nm with negative surface charge of about −12 mV. Later, hepatoprotective studies were performed by administering AMP to albino mice only once at a dosage of 2 g/kg p.o. Post 24 h of AMP intoxication, the animals were treated orally with three different doses of TiO2 NPs (150, 100, and 50 mg/kg) or silymarin at an amount of 50 mg/kg p.o., administered only once. Post 24 h of last treatment, all the animals were surrendered to death. The group of mice administered with AMP displayed a substantial raise in the serum alkaline phosphatase (560 ± 31.90), lactate dehydrogenase (167 ± 10.17), aspartate amino transferase (251 ± 14.82), alanine amino transferase (326 + 18.96), bilirubin (2.2 ± 1.066), cholesterol (96.7 ± 6.2), and albumin (6.0 ± 1.27) in serum, which signified the liver damage. A considerably depleted level of glutathione (5.4 + 1.24) was detected in the mice intoxicated with AMP. The activities of catalase (33.2 ± 2.66) and superoxide dismutase (36 ± 2.93) declined after exposure to acetaminophen, resulting in oxidative stress in liver. The performance of adenosine triphosphatase (901 ± 50.7) and glucose‐6‐phosphatase (3.9 + 1.15) were considerably inhibited after administrating with AMP. On treating with TiO2 NPs, all the variables reversed significantly towards regular levels and were noticed to be nontoxic. The groups treated with TiO2 NPs exhibited less activity of ALT, AST, LDH, and SALP in serum, indicating the protective effect of TiO2 NPs to prevent liver damage. Exposure with TiO2 NPs inverted the cholesterol, albumin, and bilirubin levels towards normal, which is similar to silymarin treatment. TiO2 NPs exhibited a substantial change in tissue and blood biochemical parameters with that of control groups. These results indicate that TiO2 NPs possesses hepatoprotective properties that mitigate the hepatotoxicity induced by acetaminophen in rats. Hence, TiO2 NPs can be further utilized in the preparation of medicine against hepatic and renal diseases, post further clinical and preclinical studies.