This study aims to investigate the impact of Perilla seed oil (PSO) on the pharmacokinetics of α-linolenic acid (ALA) in vivo, as well as its hepatoprotective effects. SD rats were selected and divided into male and female groups, with five rats in each group. The concentration of ALA in plasma at various time points was measured, and pharmacokinetic parameters were calculated using DAS 2.0 software. Concurrently, KM mice were randomly divided into five groups (n = 10). The levels of malondialdehyde (MDA), glutathione (GSH), and triglycerides (TG) in the liver were assessed using biochemical assay kits. The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were detected using a biochemical analyzer. Additionally, the serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-21 were measured by ELISA. Pathological changes in the liver tissues of mice were observed using HE staining, while changes in lipid deposition in the liver were assessed using oil red O staining. Furthermore, we established an ethanol (EtOH) injury model in human hepatoblastoma HepG2 cells. The proliferative activity of HepG2 cells was evaluated using CCK-8, and the release of ALT, AST, and lactate dehydrogenase (LDH) in the cell supernatant was measured using a biochemical analyzer. In antitumor activity assay, we employed the cell scratch assay to evaluate the migration rate of HepG2 cells and the Transwell plate method to assess the invasion capability of HepG2 cells. The results revealed significant gender differences in the pharmacokinetic parameters of ALA, with the peak concentration (C_max), area under the curve (AUC) from 0 to t, and AUC from 0 to infinity in the plasma of male mice being 10 times, 9.7 times, and 14.5 times those of female mice, respectively. However, the peak time (T_max) remained consistently at 360 minutes. In the alcoholic liver injury model, PSO significantly reduced the levels of MDA and TG in the liver, as well as the serum levels of AST, ALT, TNF-α, IL-6, and IL-21 in mice with alcoholic liver injury. Furthermore, PSO increased the level of GSH in the liver, markedly improved pathological liver tissue damage, and reduced lipid deposition. In vitro experiments confirmed that PSO could enhance the survival rate of HepG2 cells damaged by EtOH and reduce the release of ALT, AST, and LDH. In the antitumor activity research, PSO significantly inhibited the proliferation, migration, and invasion abilities of HepG2 cells. In summary, male mice exhibited significantly better absorption efficiency of ALA from PSO compared to female mice. PSO can alleviate alcoholic liver injury and protect HepG2 cells damaged by EtOH, which may be related to its anti-inflammatory and antioxidant stress effects. Additionally, PSO also exhibits potential antitumor activity.