Objective To investigate the effect of leucine carboxyl methyltransferase 1 (LCMT1) knockout on fructose-induced lipid deposition in primary mouse hepatocytes. Methods Primary hepatocytes were isolated from wild-type (WT) and hepatocyte-specific LCMT1 knockout (KO) mice via a two-step hepatic portal vein perfusion method. The cells were divided into four groups: WT-Control, WT-5 mmol/L fructose, KO-Control, and KO-5 mmol/L fructose. Cell viability was determined by Alamar-Blue. Hepatocyte injury was evaluated by measuring alanine aminotransferase and aspartate aminotransferase levels. Lipid deposition was visualized via Oil Red O staining and lipid droplet green fluorescence staining, whereas the cellular triglyceride content was quantified via a GPO-POD assay kit. The mRNA expression of lipid metabolism-related genes was detected via quantitative real-time PCR, and the protein expression of LCMT1 and PP2Ac was detected via Western blotting. Results Fructose treatment did not significantly alter cell viability in any group, and no significant cell damage was observed (P > 0.05). Compared with the WT-Control group, the WT-5 mmol/L fructose group presented marked accumulation of lipid droplets in hepatocytes (P < 0.001), with significantly elevated triglyceride content (P < 0.05). The mRNA levels of the de novo lipid synthesis genes ChREBP, SREBP-1c, and ACC1 were increased (P < 0.05, P < 0.001, P < 0.001), whereas FAS expression was not significantly altered (P > 0.05). The mRNA levels of the lipid uptake genes FABP1 and FATP2 were also significantly increased (P < 0.05, P < 0.05). In contrast, the KO-5 mmol/L fructose group presented a reduced number of lipid droplets (P < 0.01, P < 0.001), decreased triglyceride content (P < 0.05), and downregulated mRNA levels of ChREBP, SREBP-1c, ACC1, FABP1, and FATP2 (P < 0.01, P < 0.001, P < 0.001, P < 0.001, P < 0.05). In contrast, CPT1 mRNA levels were markedly increased (P < 0.01). Compared with that in the WT-Control group, the total expression of PP2Ac was significantly greater (P < 0.05), and PP2Ac demethylation was significantly lower (P < 0.01) in the WT-5 mmol/L fructose group. In the KO-Control group, total PP2Ac expression remained unchanged (P > 0.05), whereas PP2Ac demethylation was markedly elevated (P < 0.001). Compared with the WT-5 mmol/L fructose group, the KO-5 mmol/L fructose group presented markedly decreased total PP2Ac expression and significantly elevated PP2Ac demethylation levels (P < 0.05, P < 0.01). Conclusion LCMT1 knockout alleviates fructose-induced lipid deposition in primary hepatocytes by inhibiting lipid uptake, enhancing fatty acid oxidation, and downregulating de novo lipid synthesis. This mechanism involves LCMT1 knockout-mediated upregulation of PP2Ac demethylation, thereby modulating PP2A activity.