Background: Non-alcoholic fatty liver disease (NAFLD) is characterized by diffuse hepatocyte steatosis. The pathogenesis of NAFLD is not recognized. It has become apparent that genetic, environmental, and metabolic factors drive NAFLD. Moreover, earlier studies have observed that some polymorphisms raise the likelihood of NAFLD. Among these, the most robustly observed was the relationship between NAFLD risk and patatin-like phospholipase domain-containing 3 (PNPLA3). Aim: We aimed to assess the effect of the rs738409 polymorphism of the PNPLA3 gene (encoding I148m) on NAFLD. Patients and Methods: This study, which included 30 participants, was conducted with meticulous attention to detail and thoroughness: 20 patients with NAFLD and ten healthy participants as a control group. Participants were diagnosed according to a liver biopsy taken during surgery from patients who were candidates for bariatric surgery and as part of a predonation assessment for candidates for donation in liver transplantation. Genotyping of the PNPLA3 gene variant (rs738409 C/G) was assessed using the TaqMan assay quantitative polymerase chain reaction in blood cells. Results: Considering the distribution of the PNPLA3 gene variant (rs738409 C/G), a greater prevalence of heterozygous rs738409 CG and homozygous rs738409 GG variants occurred in the NAFLD patients in contrast to the control group (p = 0.048). In addition, NAFLD patients with the homozygous GG variant had a higher incidence of hepatic fibrosis (p = 0.018). Furthermore, PNPLA3 gene polymorphism significantly predicted hepatic steatosis and fibrosis in NAFLD patients (p = 0.033 and p < 0.001, respectively). Conclusion: The PNPLA3 rs738409 polymorphism is significantly associated with the susceptibility and severity of non-alcoholic fatty liver disease.