© 2018 Alexander et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background and purpose Liver disease, particularly non-alcoholic fatty liver disease (NAFLD), is a risk factor for cardiovascular disease, but little is known about its relationship with ischemic stroke. Methods In the Reasons for Geographic and Racial Differences in Stroke (REGARDS) cohort of 30,239 American black and white adults, we assessed baseline NAFLD as fatty liver index (FLI) >60, and assessed liver biomarkers aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (GGT), and the AST/ALT ratio and risk of incident ischemic stroke over 5.8 years using a case-cohort study design. Results Considering 572 strokes and a 1,017-person cohort sample, NAFLD was inversely associated with stroke risk in men (HR: 0.50; 95% CI: 0.26, 0.96), as was being in the highest ALT quintile versus the lowest (HR: 0.39; 95% CI: 0.19, 0.78) and the highest versus lowest GGT quintile (HR: 0.45, 95% CI: 0.24, 0.85), but not in women. Conversely, FLI score above the 90th percentile was associated with increased stroke risk among women (HR: 2.26; 95% CI: 1.14–4.47), but not men. AST was not associated with stroke risk in either sex. AST/ALT ratio >2 was strongly associated with increased stroke risk in whites, but not blacks (HRs: 3.64; 95% CI: 1.42–9.35 and 0.97; 95% CI: 0.45–1.99, respectively; p for interaction = 0.03). Conclusions The relationships between NAFLD, liver biomarkers, and ischemic stroke are complex, and sex and race differences we observed require further study and confirmation.