Abstract Background We have previously shown the value of next-generation des-r-carboxy prothrombin (NX-DCP) for predicting vascular invasion in hepatocellular carcinoma (HCC). Since conventional DCP is inaccurate under some conditions, this study aimed to assess whether NX-DCP immunohistochemical staining was related to vascular invasion in HCC. Methods Fifty-six patients scheduled to undergo resection for single HCC were divided into two groups, with and without pathological portal vein invasion. Immunohistochemical features of HCC and sites of vascular invasion were assessed using alpha-fetoprotein (AFP), conventional DCP, and NX-DCP. Results Pathological portal vein invasion was absent in 43 patients and present in 13 patients. Patient characteristics, pathological background of the liver parenchyma, and tumor-related factors did not differ significantly between the groups. There was no significant difference in the serum AFP level between the groups, whereas levels of conventional DCP (p < 0.0001) and NX-DCP (p < 0.0001) were significantly higher in the vascular invasion group. Immunohistochemical staining showed no significant difference in the staining rate of tumor (67.9% vs. 80.7%, p = 0.08), but NX-DCP stained significantly more at the sites of vascular invasion (15.4% vs. 46.2%, p = 0.01) than conventional DCP. No vascular invasion was stained by AFP. Conclusions NX-DCP offers better sensitivity for detecting sites of vascular invasion than AFP and conventional DCP.