To investigate the effect of polyene phosphatidyl choline (PPC) on hepatocyte steatosis and the possible role of the peroxisome proliferators-activated receptor α (PPARα)/carnitine palmityl transferase 1A (CPT-1A) pathway in the treatment. L02 hepatocyte steatosis was induced with PPC treatment, and Oil Red O staining and triglyceride measurement were used to evaluate the influence of PPC on the degree of steatosis. Quantitative PCR and Western blot were used to measure the changes in mRNA and protein of CPT-1A, followed by using GW6471 to specifically inhibit the PPARα pathway. The changes in the above indices were observed, and one-way analysis of variance and LSD method for pairwise comparison were used to analyze the data. Compared with the blank control and DMSO group, the PPC treatment group had a significantly lower triglyceride content after steatosis (214.97±25.53 and 219.62±19.40 μg/mg vs 163.82±14.94 μg/mg, F = 6.90, P < 0.05), but had significantly higher levels of mRNA (0.36±0.04 and 0.37±0.04 vs 0.75±0.09, F = 38.37, P < 0.05) and protein of CPT-1A. After inhibition by GW6471, a specific PPARα inhibitor, the content of triglyceride showed no significant difference between the blank control group, DMSO group, and PPC treatment group (244.04±22.38 μg/mg vs 242.27±18.71 μg/mg vs 225.41±27.63 μg/mg, F = 0.59, P > 0.05), and the levels of mRNA (0.16±0.06 vs 0.17±0.02 vs 0.18±0.04, F = 0.18, P > 0.05) and protein of CPT-1A also showed no significant difference between the three groups. PPC may relieve hepatocyte steatosis through activation of the PPARα/CPT-1A pathway.