As a broad-spectrum antibiotic, gentamicin is used extensively in T cell culturing in vitro, but preliminary studies have identified that T cell activity is significantly affected by gentamicin. In the present study, the hemocyanin from Litopenaeus vannamei (L. vannamei) was selected as an additive for T cell cultures in vitro. Compared with those in the control group, the cell quantity exhibited no significant difference, and the formation rate of cell colony increased gradually with increases in the hemocyanin concentration. Additionally, flow cytometry assays identified that cluster of differentiation (CD)3+CD4+ and CD4+CD25+ T cells in the 0.2 µg/ml hemocyanin (Hem 3) group were all significantly increased. Furthermore, cell cycle analysis demonstrated that T cells in the G0/G1 phase were significantly decreased in the Hem 3 group compared with in the control, 0.05 µg/ml (Hem 1) and 0.1 µg/ml (Hem 2) groups, and cells in the S phase were significantly elevated in the Hem 3 group compared with in the control and Hem 1 groups. In addition, MTT analysis indicated that the cytotoxicity of T cells towards HepG2 cells was significantly increased in the Hem 3 group compared with in the control, Hem 1 and Hem 2 groups. Taken together, the present study identified that hemocyanin may improve the proliferation and cytotoxicity of T cells, and the results supported the use of hemocyanin in T cell adoptive immunotherapy.