Psychological stress may have adverse metabolic effects and induce unhealthy behaviors, but the role of stress in the development of non-alcoholic fatty liver disease (NAFLD) is largely unexplored. We investigated the association between perceived stress and the prevalence of NAFLD in a large sample of apparently healthy men and women. We performed a cross-sectional study of 171,321 adults who underwent health screening examination between 2011 and 2013 in one health screening center. Perceived stress was assessed using the short version of the Perceived Stress Inventory (PSI). NAFLD was assessed using ultrasonography in the absence of excessive alcohol use or any other identifiable cause of liver disease. The prevalence of NAFLD was 27.8%. In fully-adjusted multivariable models, the odds ratio (95% confidence intervals) for NAFLD comparing participants in the 5th quintile of PSI score (≥23) with those in the lowest quintile (<12) was 1.17 (1.11, 1.22), with a moderately increased prevalence of NALFD across quintiles of PSI score. The positive association between PSI score and NAFLD was observed in all subgroups analyzed, although the association was stronger in men compared to women (p interaction <0.001), and in obese compared to non-obese (p interaction 0.005). In this large study of apparently healthy men and women, higher perceived stress was independently associated with an increased prevalence of NAFLD, supporting a possible relationship between perceived stress and NAFLD. Prospective study is needed to elucidate mediating mechanisms to warrant stress management to reduce NAFLD.