AbstractBovine parainfluenza virus type 3 (BPIV3) is a viral respiratory pathogen that infects cattle and causes significant economic losses. We generated a recombinant adenovirus called rHAd5-F + HN by expressing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein of BPIV3 using the human adenovirus serotype 5 (rHAd5). We evaluated its effects on humoral and cellular immune responses in mice (n = 45) and calves (n = 9). Serum antibody responses were assessed by enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HI), and neutralising antibodies (NAb). After boosting immunity with rHAd5-F + HN, mice produced significantly higher levels of antibodies against the BPIV3 genotype A and genotype C strains. The production of antibodies exceeded those produced by adenoviruses rHAd5-F and rHAd5-HN, which express the F and HN glycoprotein, respectively. The percentages of splenic CD3+/CD8+T lymphocytes and IL-4+ cytokines in rHAd5-F + HN mice were considerably higher than those in the control group. Mice immunised with rHAd5-F + HN exhibited much lower viral loads in the lungs and tracheas compared to the control group. Additionally, the lungs of mice vaccinated with rHAd5-F + HN showed no notable histopathological changes. On the other hand, rHAd5-F + HN produced a humoral immune response in calves. Following the booster intramuscular injection with the rHAd5-F + HN, the serum antibody levels against BPIV3 genotype C strain were 1:20 452, 1:1024, and 1:426 in calves, as detected by ELISA, HI, and NAb, respectively. The HI and NAb levels against the BPIV3 genotype A strain were 1:213 and 1:85 in calves, respectively. These results indicate that rHAd5-F + HN effectively induced immunity against BPIV3 infection.