BACKGROUND The adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1 (APPL1) has been suggested to play an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). The aim of this article is to explore the relationship between APPL1 rs4640525 polymorphism and the susceptibility of NAFLD in a Chinese Han population. METHODS In this study, rs4640525, a significant single nucleotide polymorphism, in the APPL1 gene was detected by the polymerase chain reaction-restriction fragment length polymorphism method, followed by extracting genomic DNA from peripheral blood leukocytes from patients with NAFLD (n = 280) and matched controls (n = 281). RESULTS The frequency of both the GG genotype and the G carrier (CG+GG) genotype were higher in NAFLD subjects compared with control subjects (all p < 0.05). The G allele frequency was 0.3036 in the NAFLD group and 0.2206 in the control group, showing a significant difference (p < 0.05). What is more, multivariate logistic regression analysis suggested that the GG genotype, G carrier genotype, body mass index, waist circumference, white blood cells, total cholesterol, alanine aminotransferase, and γ-glutamyl-transferase might be associated with an increased susceptibility of NAFLD (all p < 0.05). CONCLUSIONS The article provides evidence that GG genotype and G carrier (CG+GG) genotypes of the rs4640525 polymorphism in the APPL1 gene may be suitable susceptibility biomarkers for NAFLD.