Carbon tetrachloride (CCl₄) is a well-known hepatotoxic chemical. Exposure to CCl₄ produces free radicals, which induce oxidative stress and cause hepatic injury. We demonstrated previously that pretreatment with zinc (Zn), which induces metallothionein (MT) expression, prevents CCl₄-induced lethal toxicity in a dose-dependent manner. While MT has been suggested as a possible hepatoprotective protein, its mechanism of protection remains unknown. In the current study, we evaluated the protective mechanism of MT, an endogenous scavenger of free radicals, against CCl₄-induced toxicity through subcutaneous administration of 50 mg/kg Zn (as ZnSO4) once daily for three consecutive days, prior to a single intraperitoneal injection of 4 g/kg CCl₄ in male ddY mice. Our results showed that Zn pretreatment significantly decreased aspartate aminotransferase and total cholesterol levels, 6-hr after CCl₄ injection, as well as lipid peroxidation. Moreover, CCl₄-induced hepatic calcium level was downregulated by pretreatment with Zn while Zn-induced MT expression decreased by more than 500 μg/g liver (43%) in the Zn + CCl₄-treated group, implying that MT was consumed by CCl₄-induced free radicals. These findings suggest that prophylaxis with Zn protects mice from CCl₄-induced acute hepatotoxicity, presumably by inducing the expression of free radical-scavenging MT.