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 group, WT-fructose group, KO-control group, and KOfructose group. Cell viability was determined through Alamar-Blue assays. Hepatocyte injury was evaluated based on alanine aminotransferase and aspartate aminotransferase levels. Lipid deposition was visualized via Oil Red O staining and lipid droplet green fluorescence staining, and the cellular triglyceride content was quantified via a GPO-POD assay. 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 blot. Results Fructose treatment did not alter cell viability significantly in any group, and no significant cell damage was observed (P>0.05). The WT-fructose group exhibited greater accumulation of lipid droplets in hepatocytes than that in the WT-control group (P<0.001), with significantly elevated triglyceride contents (P<0.05). The mRNA levels of the de novo lipid synthesis genes ChREBP, SREBP-1c, and ACC1 were increased significantly (P<0.05, P<0.001, P<0.001), whereas FAS expression did not differ significantly between groups (P>0.05). The mRNA levels of the lipid uptake genes FABP1 and FATP2 also increased significantly (both P<0.05). In contrast, the KO-fructose group presented a reduced number of lipid droplets (P<0.01, P<0.001), decreased triglyceride content (P<0.05), and decreased 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); CPT1 mRNA levels were markedly increased (P<0.01). Total PP2Ac expression was significantly higher (P<0.05) and PP2Ac demethylation was significantly lower (P<0.01) in the WT-fructose group than in the WT-control 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 levels in the WT-fructose group, the KO-fructose group presented markedly lower total PP2Ac expression and significantly higher PP2Ac demethylation levels (P<0.05, P<0.01, respectively). Conclusions LCMT1 knockout alleviates fructose-induced lipid deposition in primary hepatocytes by inhibiting lipid uptake, increasing fatty acid oxidation, and downregulating de novo lipid synthesis. These effects are medicated by the LCMT1 knockout-mediated upregulation of PP2Ac demethylation, thereby modulating PP2A activity.