BACKGROUND Serum gamma-glutamyl transferase (GGT) is a marker of oxidative stress, associated with increased cardiovascular (CV) risk. The impact of smoking on oxidative stress may be aggravated in individuals with non-alcoholic fatty liver disease (NAFLD). We aimed to ascertain the association of smoking on GGT levels in the presence or absence of NAFLD. METHODS We evaluated 6,354 healthy subjects (43 ± 10 years, 79% males) without clinical cardiovascular disease (CVD) undergoing an employer-sponsored physical between December 2008 and December 2010. NAFLD was diagnosed by ultrasound and participants were categorized as current or non-smokers by self report. A multivariate linear regression of the cross-sectional association between smoking and GGT was conducted based on NAFLD status. RESULTS The prevalence of NAFLD was 36% (n = 2,299) and 564 (9%) were current smokers. Smokers had significantly higher GGT levels in the presence of NAFLD (P < 0.001). After multivariable adjustment, current smoking was associated with 4.65 IU/L higher GGT level, P < 0.001, compared to non-smokers. When stratified by NAFLD, the magnitude of this association was higher in subjects with NAFLD (β-coefficient: 11.12; 95% confidence interval (CI): 5.76 - 16.48; P < 0.001); however, no such relationship was observed in those without NAFLD (β: -0.02; 95% CI: -3.59, 3.56; P = 0.992). Overall the interaction of NAFLD and smoking with GGT levels as markers of oxidative stress was statistically significant. CONCLUSIONS Smoking is independently associated with significantly increased oxidative stress as measured by GGT level. This association demonstrates effect modification by NAFLD status, suggesting that smoking may intensify CV risk in individuals with NAFLD.