Objective <p>The objective of this study was to investigate the effect of silibinin (SLB) on CAT, GSH-Px, and SOD enzyme activities, caspase-3, COX-2, and anti-eNOS expression cobalt (Co)-induced hepatotoxicity in rats.</p> Methods <p>Male Wistar albino rats (n = 24) were randomly divided into four groups: the control (0.5&#xa0;mL of isotonic solution), Co (150&#xa0;mg/kg/day), SLB (100&#xa0;mg/kg/day), and Co + SLB (150&#xa0;mg/kg/day + 100&#xa0;mg/kg/day). The experimental design was carried out as a randomized complete block design.</p> Results <p>The catalase enzyme activity decreased to 3.55 EU/mg of protein in the Co group, while it was 19.82 EU/mg of protein in the control group (<i>p</i> &lt; 0.01). In the SLB group rat liver CAT enzyme activity increased compared with the Co group (<i>p</i> &lt; 0.01). According to the results of statistical analysis, a significant difference (<i>p</i> &lt; 0.01) in glutathione peroxidase enzyme activity was observed between the control group and Co group. However, no statistically significant difference (<i>p</i> &gt; 0.05) was observed between the Co + SLB group compared to the Co group (<i>p </i>˃ 0.05). In addition, no significant difference (<i>p</i> &gt; 0.05) in superoxide dismutase enzyme activity between control and Co + SLB group groups was detected. Histopathological examination revealed marked hepatic damage in the Co group, whereas structural improvement was observed in the Co + SLB group. Immunohistochemical evaluation demonstrated increased caspase-3 and COX-2 immunoreactivity in the Co group compared to the control group. In the Co + SLB group, immunoreactivity for caspase-3 and COX-2 appeared reduced, while eNOS expression appeared more prominent compared to the Co group.</p> Conclusion <p>These results showed that SLB provides biochemical, histopathological, and immunohistochemical protection against Co-induced liver toxicity.</p>