Previously, we reported that inverse associations of high-density lipoprotein (HDL) with cardiovascular disease and diabetes were only observed for HDL that lacked the pro-inflammatory protein apolipoprotein C3 (apoC3). To provide further insight into the cardiometabolic properties of HDL subspecies defined by the presence or absence of apoC3, we aimed to examine these subspecies with liver fat content and non-alcoholic fatty liver disease (NAFLD). We investigated cross-sectional associations between ELISA-measured plasma levels of apoA1 in HDL that contained or lacked apoC3 and computed tomography-determined liver fat content and NAFLD (14 drinks/week in men and >7 drinks/week in women). In multivariable-adjusted regression models, apoA1 in HDL that contained or lacked apoC3 was differentially associated with liver fat content (P-heterogeneity = 0.048). While apoA1 in HDL that lacked apoC3 was inversely associated with liver fat content (P-trend <0.0001), apoA1 in HDL that contained apoC3 was not statistically significantly associated with liver fat content (P-trend = 0.57). Higher apoA1 in HDL that lacked apoC3 was related to a lower prevalence of NAFLD (OR per SD: 0.80; 95% CI: 0.72, 0.89), whereas no association was found for apoA1 in HDL that contained apoC3 (OR per SD: 0.95; 95% CI: 0.85, 1.05; P-heterogeneity = 0.09). Higher apoA1 in HDL that lacked apoC3 was associated with less liver fat content and a lower prevalence of NAFLD. This finding extends the inverse association of HDL lacking apoC3 from cardiovascular disease to NAFLD. Lack of biopsy-proven hepatic steatosis and fibrosis data requires the replication of our study in further studies.