Introduction Bovine adenovirus (BAdV) type 3 causes respiratory and gastroenteric diseases of varying severity in cattle, particularly newborn calves. Trials have been conducted of a vaccination against the diseases caused by BAdV using both modified live-virus and inactivated-virus preparations in cattle, but no commercial BAdV-3 vaccine has yet reached the market. Therefore, there is an urgent need to develop new, safe, and effective vaccines against BAdV-3. Material and Methods Recombinant hexon protein (rhexon) of BAdV-3 was expressed in the E. coli system to evaluate immune response in mice and goats. Antibody responses and cytokine levels were analysed and the effects of administrations of different amounts of recombinant protein compared. Long-term antibody production was evaluated by indirect ELISA, and the total immunoglobulin G secreted by goats and mice immunised with the purified rhexon protein was determined. Results The immunised mice had a stronger antibody response than the control group at eight weeks post vaccination. The immunised groups also showed significantly higher (P ˂ 0.05) expression of interferon-γ, interleukin 2 (in mice), and interleukin 21 (in goats) at four weeks. Furthermore, vaccination with rhexon was able to induce long-term antibody production for at least 16 weeks in mice and goats. Conclusion The rhexon protein induced immune responses, especially long-term antibody production and T helper 1 cell cytokine production in mice and goats. The immunogenic properties of this protein make it a promising subunit vaccine antigen.