BackgroundNon-alcoholic fatty liver disease (NAFLD) shares common risk factors with cardiovascular diseases. Effects of longitudinal trends in non-high-density lipoprotein (non-HDL) cholesterol on NAFLD development are not understood. This study aimed to assess the relationship between non-HDL cholesterol trajectories and the incidence of NAFLD and to identify genetic differences contributing to NAFLD development between non-HDL cholesterol trajectory groups.MethodsWe analyzed data from 2203 adults (aged 40–69 years) who participated in the Korean Genome and Epidemiology Study. During the 6-year exposure periods, participants were classified into an increasing non-HDL cholesterol trajectory group (n= 934) or a stable group (n= 1269). NAFLD was defined using a NAFLD-liver fat score > -0.640. Multiple Cox proportional hazard regression analysis estimated the hazard ratio (HR) and the 95% confidence interval (CI) for the incidence of NAFLD in the increasing group compared with the stable group.ResultsA genome-wide association study identified significant single-nucleotide polymorphisms (SNPs) associated with NAFLD. During the median 7.8-year of event accrual period, 666 (30.2%) newly developed NAFLD cases were collected. Compared with the stable non-HDL group, the adjusted HR (95% CI) for the incidence of NAFLD in the increasing non-HDL cholesterol group was 1.46 (1.25–1.71). Although there were no significant SNPs, the polygenic risk score was highest in the increasing group, followed by the stable and control groups.ConclusionOur study indicates that lifestyle or environmental factors have a greater effect size than genetic factors in NAFLD progression risk. Lifestyle modification could be an effective prevention strategy for NAFLD for people with elevated non-HDL cholesterol.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-023-04291-4.