Summary Background Patients with nonalcoholic steatohepatitis (NASH) have gut dysbiosis and intestinal bacterial overgrowth. Aim To test the hypothesis that endotoxemia is associated with the histological severity of nonalcoholic fatty liver disease (NAFLD) and determine factors associated with endotoxemia. Methods The endotoxemia markers lipopolysaccharide‐binding protein (LBP) and endotoxin levels were measured in 237 NAFLD patients 1 day before liver biopsy. Biomarkers of liver injury and transient elastography were performed as additional markers of disease severity. Results A total of 114/237 (48%) patients had NASH and 80/237 (34%) had F2‐4 fibrosis. LBP was correlated with lobular inflammation (P=.001), while both LBP (P=.0004) and endotoxin levels (P=0.008) were correlated with fibrosis. LBP was also correlated with cytokeratin‐18 fragments (P=.002) and aspartate aminotransferase‐to‐alanine aminotransferase ratio (P=.006), and both LBP (P=.019) and endotoxin (P=.006) were correlated with liver stiffness measurement by transient elastography. LBP was increased in patients with NASH (15.3±4.6 vs 13.8±3.3 μg/mL; P=.005) and F2‐4 fibrosis (15.4±4.4 vs 14.0±3.7 μg/mL; P=.008). Interestingly, patients harbouring the TM6SF2 rs58542926 T allele that predispose to NAFLD/NASH had higher LBP level. By multivariate analysis, gender, higher body mass index and glycated haemoglobin, and TM6SF2 variants were independent factors associated with increased LBP level. Conclusions Endotoxemia is positively associated with NASH and significant fibrosis. The association between TM6SF2 and endotoxemia warrants further investigations. The findings may shed light on the pathogenesis of NASH and inform a novel treatment target. Linked ContentThis article is linked to Valenti and Romeo paper. To view this article visit https://doi.org/10.1111/apt.14154.