To assess the diagnostic performance of the H-score of 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in the histopathological diagnosis of adrenocortical tumors (ACT). We retrospectively evaluated 199 cases of ACT, of which 85 were diagnosed as aldosterone-producing adenoma (APA), 66 as cortisol-producing adenoma (CPA), 9 as aldosterone-cortisol co-secreting adenoma, 30 as nonhyperfunctioning adenoma, and 9 as adrenocortical carcinoma (ACC). Immunohistochemical staining was performed using anti-CYP11B1 and anti-CYP11B2 monoclonal antibodies. The staining was quantified by the McCarty's H-score system. The diagnostic performance was assessed by the receiver operating characteristic curve (ROC). The H-score of CYP11B1 is highest in the CPA group and lowest in the ACC group. The H-score of CYP11B2 in the APA group is significantly higher than other ACT groups. The area under ROC (AUC) of an increased H-score of CYP11B2 (>65) for the diagnosis of APA was 0.971 (95%CI 0.937-0.990). The AUC of an increased H-score of CYP11B1 (>204) for the diagnosis of CPA was 0.725 (95%CI 0.658-0.786). The AUC of a decreased H-score of CYP11B1 (<85) for the diagnosis of ACC was 0.960 (95%CI 0.923-0.983). H-score of CYP11B1 and CYP11B2 are reliable tools for the histopathological subtyping of functional benign ACT and may offer some value in the histopathological diagnosis of malignant ACT.