We aimed to assess liver histological changes of HBeAg-negative chronic hepatitis B (CHB) patients with normal ALT, and determined the association between significant liver injury and age, ALT, and HBV DNA levels. We retrospectively examined 327 patients who underwent liver biopsy from 2009 to 2018. Significant liver histological change is defined as liver necroinflammation ≥ G2 and/or liver fibrosis ≥ F2. The proportion of patients with significant liver necroinflammation or fibrosis in the high-normal ALT group (ALT > 20 U/L) was higher than that in the low-normal ALT group (ALT ≤ 20 U/L) (44.6% vs 26.5%, 61.0% vs 41.7%, p 20 U/L and HBV DNA ≥ 2000 IU/mL were independently associated with significant liver histopathology (p 20 U/L and HBV DNA ≥ 2000 IU/mL are good independently predictive factors for evaluating significant liver histopathology for HBeAg-negative CHB patients with normal ALT. Chinese Clinical Trial Registry (ChiCTR-IOR-14005474).