Background. The aim of this study was to provide new insights into the prevalence of positive antinuclear antibody (ANA) in patients with HBV-related acute-on-chronic liver failure (ACLF) and its impact on clinical outcomes. Methods. A total of 116 patients with HBV-related ACLF treated at three clinical centers were retrospectively recruited. Serum concentrations of ANA were detected using the enzyme-linked immunosorbent assay kit. Multiple nuclear dots, rim-like, and centromere patterns of ANA were detected using indirect immunofluorescence assay on HEp-2 cells. Results. Among the 116 patients with HBV-related ACLF, 17 (14.66%) were ANA positive. Most patients in both ANA positive and negative groups were males (88.2% and 83.8%). Patients with negative ANA had a higher international normalized ratio, model for end-stage liver disease (MELD), and MELD-sodium scores than those with positive ANA (all P < 0.05). Multiple nuclear dot pattern was detected in half of the patients (8/17, 47.06%), rim-like/membranous pattern was found in six patients, and centromere pattern was detected in the last three patients. For patients with ANA (+), IgM was lower, and it was positively correlated with IgG. For patients with ANA (-), C3 was positively correlated with C4, and both C3 and C4 were negatively correlated with INR and MELD (all P < 0.05). In addition, TBIL, INR, WBC, and PLT, but not ANA, resulted as independent risk factors associated with 90-day mortality. Conclusion. Positive ANA is frequent in HBV-related ACLF, and it does not seem to be associated with poor outcomes, but the pathogenesis of ACLF may be different between ANA (+) and ANA (-) groups.