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Objective: Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. The identification of new simple, inexpensive and highly accurate markers for HCC diagnosis and screening is needed. This case-control study evaluates the role of annexin A2 and voltage-gated calcium channels α2δ1 subunit as serum biomarkers for HCC diagnosis. Methods: The study comprised three groups: group 1, 50 patients with an initial diagnosis of HCC associated with chronic hepatitis C virus infection; group 2, 25 patients diagnosed with chronic hepatitis C virus infection and cirrhosis without any evidence of HCC; and group 3, 15 healthy controls. All participants were subjected to clinical and laboratory investigations, and radiological scanning. The serum levels of alpha-fetoprotein (AFP), annexin A2, and the α2δ1 subunit were evaluated by using ELISA technique. Results: The serum levels of annexin A2 significantly increased in patients with HCC (10.4±2.5 ng/mL; P<0.001) or with cirrhosis (9.31±1.8 ng/mL; P<0.001) comparing to that of healthy controls (0.296±0.09 ng/mL). However, there was no significant difference in serum annexin A2 levels in patients with HCC comparing to those with cirrhosis. Serum α2δ1 subunit significantly increased in patients with HCC (20.12±3.7 ng/mL) comparing to that in patients with cirrhosis (10.41±3.4 ng/mL, P<0.001) and healthy controls (10.2±2.9 ng/mL, P<0.001). Conclusions: The serum α2δ1 subunit may function as a new biomarker for HCC diagnosis. Conversely, serum annexin A2 has low diagnostic value as an HCC marker, especially in patients with underlying cirrhosis.