| Summary: | (1) Background: the current research was conducted to investigate the potential non-antioxidant roles of vitamin E in the protection of hepatocysts from oxidative damage. (2) Methods: primary sheep hepatocytes were cultured and exposed to 200, 400, 600, or 800 μmol/L hydrogen peroxide, while their viability was assessed using a CCK-8 kit. Then, cells were treated with 400 μmol/L hydrogen peroxide following a pretreatment with 50, 100, 200, 400, and 800 μmol/L vitamin E and their intracellular ROS levels were determined by means of the DCF-DA assay. RNA-seq, verified by qRT-PCR, was conducted thereafter: non-treated control (C1); cells treated with 400 μmol/L hydrogen peroxide (C2); and C2 plus a pretreatment with 100 μmol/L vitamin E (T1). (3) Results: the 200–800 μmol/L hydrogen peroxide caused significant cell death, while 50, 100, and 200 μmol/L vitamin E pretreatment significantly improved the survival rate of hepatocytes. ROS content in the cells pretreated with vitamin E was significantly lower than that in the control group and hydrogen-peroxide-treated group, especially in those pretreated with 100 μmol/L vitamin E. The differentially expressed genes (DEGs) concerning cell death involved in apoptosis (<i>RIPK1</i>, <i>TLR7</i>, <i>CASP8</i>, and <i>CASP8AP2</i>), pyroptosis (<i>NLRP3</i>, <i>IL-1β</i>, and <i>IRAK2</i>), and ferroptosis (<i>TFRC</i> and <i>PTGS2</i>). The abundances of <i>IL-1β</i>, <i>IRAK2</i>, <i>NLRP3</i>, <i>CASP8</i>, <i>CASP8AP2</i>, <i>RIPK1</i>, and <i>TLR7</i> were significantly increased in the C1 group and decreased in T1 group, while <i>TFRC and PTGS2</i> were increased in T1 group. (4) Conclusions: oxidative stress induced by hydrogen peroxide caused cellular damage and death in sheep hepatocytes. Pretreatment with vitamin E effectively reduced intracellular ROS levels and protected the hepatocytes from cell death by regulating gene expression associated with apoptosis (<i>RIPK1</i>, <i>TLR7</i>, <i>CASP8</i>, and <i>CASP8AP2)</i> and pyroptosis (<i>NLRP3</i>, <i>IL-1β</i>, and <i>IRAK2</i>), but not ferroptosis (<i>TFRC and PTGS2)</i>.
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