Objective To investigate the prevalence and neutralizing activity of anti-human adenovirus type 55 (anti-HAdV55) among the soldiers in Southwest China, so as to provide reference for the prevention and control of HAdV55. Methods The sera of 325 soldiers in Southwest China were collected, and the anti-HAdV55 in serum was detected by ELISA. The neutralization activity of antibody positive serum was detected by neutralization test in vitro. Results The positive rate of anti-HAdV55 was low (5.85%, 19/325) among the soldiers in Southwest China. Of the 19 cases of positive serum, 14 cases were able to neutralize HAdV55 infection in human laryngeal epithelial carcinoma cell line (Hep-2 cells) with the highest titer being 1:64. Conclusion The positive rate of anti-HAdV55 is low among the soldiers in Southwest China, which suggests the soldiers in Southwest China have a low resistance to HAdV55 infection. Once the individual infection is found, the troops should strengthen the prevention and control of HAdV55 infection to prevent the spreading of infection.