Hepatocellular carcinoma (HCC), accounting for ~90% of all primary liver cancer, is a prevalent malignancy worldwide. The intratumor heterogeneity of its causative etiology, histology, molecular landscape, and immune phenotype makes it difficult to precisely recognize individuals with high mortality risk or tumor-intrinsic treatment resistance, especially immunotherapy. Herein, we comprehensively evaluated the activities of cancer hallmark gene sets and their correlations with the prognosis of HCC patients using gene set variation analysis (GSVA) and identified two HCC subtypes with distinct prognostic outcomes. Based on these subtypes, seven immune-related genes (TMPRSS6, SPP1, S100A9, EPO, BIRC5, PLXNA1, and CDK4) were used to construct a novel prognostic gene signature [hallmark-guided subtypes-based immunologic signature (HGSIS)] via multiple statistical approaches. The HGSIS-integrated nomogram suggested an enhanced predictive performance. Interestingly, oncogenic hallmark pathways were significantly enriched in the high-risk group and positively associated with the risk score. Distinct mutational landscapes and immune profiles were observed between different risk groups. Moreover, immunophenoscore (IPS) and tumor immune dysfunction and exclusion (TIDE) analysis showed different sensitivities of HGSIS risk groups for immune therapy efficacy, and the pRRophetic algorithm indicated distinguishable responses for targeted/chemotherapies in different groups. KIF2C was picked out as the key target concerning HGSIS, and the top 10 small molecules were predicted to bind to the active site of KIF2C via molecular docking, which might be further used for candidate drug discovery of HCC. Taken together, our study offers novel insights for clinically significant subtype recognition, and the proposed signature may be a helpful guide for clinicians to improve the treatment regimens.Copyright © 2022 Guo, Tang, Yang, Li and Zhang.