Thymosin alpha-1 (Tα1) has been shown to be effective in chronic hepatitis B treatment. This study investigated the effect of Tα1 and interferon-alpha (IFNα) on cytokine production by peripheral blood mononuclear cells (PBMCs) of 12 patients with eAg-negative chronic hepatitis B (HBV). We evaluated the effect of incubation with Tα1, IFNα or both on the synthesis of T-helper 1 (Th1) cytokines [interleukin-2 (IL-2), IFNγ] and Th2 cytokines (IL-4, IL-10) and of antiviral protein 2′,5′-oligoadenylate synthetase (2′,5′-OAS) in patients and in a group of 10 healthy controls. Concerning Th1 profile, controls showed lower IL-2 synthesis than HBV patients. In HBV setting, IFNα/Tα1 combination was able to increase IL-2 production significantly, when compared with baseline condition. About the Th2-cytokines, controls showed statistically lower synthesis of IL-4 and higher production of IL-10, than HBV patients. In these latter, IFNα increased the synthesis of IL-10 compared with baseline. Interestingly, both Tα1 alone and the IFNα/Tα1 combination reversed this effect. Finally, compared with baseline, the synthesis of 2′,5′-OAS was significantly higher in the presence of Tα1 and IFNα alone, and in the presence of IFNα/Tα1 association, while no differences were found between controls and HBV patients. In conclusion, in PBMCs from eAg-negative HBV patients, Tα1 alone was able to increase the antiviral protein synthesis, while in association with IFNα, it stimulated the IL-2 synthesis and inhibited the IFN-induced IL-10 production. These results need further investigations, but reinforce the idea of an immunotherapeutic approach for chronic hepatitis B.