Background: In this retrospective study, we investigated clinicopathological features and prognosis of hepatocellular carcinoma (HCC) patients applying an immunosubtyping method based on the serum alpha-fetoprotein (AFP) levels and glypican-3 (GPC3) immunohistochemical expression. Methods: Two hundred and twenty-nine HCC patients, who had been subjected to hepatectomy and accepted serum AFP and GPC immunohistochemical expression tests, were divided into four groups: AFP(+)GPC3(+), AFP(+)GPC3(-), AFP(-)GPC3(+), and AFP(-)GPC3(-) groups. During the study, HCC patients' sex ratios, ages, incidence of cirrhosis, clinicopathological features-such as tumor lesion, tumor size, histological grade, vascular invasion, regional lymph node involvement, distant metastasis-and their follow-up time were observed and continuously recorded. Results: Regarding their clinicopathological features, the four groups only differed in the histological grade with statistical significance. Furthermore, the four subtypes showed statistically significant differences in sex ratios and incidence of cirrhosis. Among the four subtypes, the prognosis was just statistically different between the AFP(+)GPC3(+) and AFP(-)GPC3(+) groups, and AFP(-)GPC3(+) was associated with a better prognosis. Conclusions: A new HCC subtype could guide the personalized therapy of HCC patients to a certain extent. The four different subtypes resulting from the AFP- and GPC3-based subclassification of HCC, especially for AFP(+)GPC3(+) and AFP(-)GPC3(-) groups, were meaningful prognostic markers for HCC.