The study aimed to determine the association of additional N-glycosylation mutations in the major hydrophilic region (MHR) of hepatitis B virus (HBV) S gene with hepatocellular carcinoma (HCC) occurrence in HBsAg/anti-HBs coexistent patients. A total of 288 HBsAg/anti-HBs coexistent patients and 490 single HBsAg-positive patients were enrolled, including 193 with HCC, 433 with chronic hepatitis B (CHB), and 152 with acute-on-chronic liver failure (ACLF). The HBV S genes were amplified from serum and sequenced. The frequency of additional N-glycosylation mutations was significantly higher in HCC patients (12.37%) than in CHB patients (4.39%) and ACLF patients (2.63%). The frequency escalated by an order of single HBsAg-positive non-HCC (1.61%), single HBsAg-positive HCC (5.98%), HBsAg/anti-HBs coexistent non-HCC (8.01%), and HBsAg/anti-HBs coexistent HCC (22.36%). Twelve kinds of mutations/mutation patterns were detected, five of which have not been reported. Multivariate analysis showed that age > 40 years [, 3.005; 95% CI, 1.177-7.674; = 0.021], alpha-fetoprotein > 10 ng/mL [, 4.718; 95% CI, 2.406-9.251; <0.001], cirrhosis [, 6.844; 95% CI, 2.773-16.891, < 0.001], Hepatitis B e antigen negativity [, 2.218; 95% CI, 4.335, = 0.020], and additional N-glycosylation mutation [, 2.831; 95% CI, 1.157-6.929; = 0.023] were independent risk factors for HCC in HBsAg/anti-HBs coexistent patients. Dynamical analysis showed that the additional N-glycosylation mutations existed 1-4 years prior to HCC occurrence in eight of 18 patients observed. In conclusion, the dditional N-glycosylation mutations together with HBsAg/anti-HBs coexistence might serve as a predictive indicator for HCC occurrence in chronic HBV-infected patients.