Mycoplasma pulmonis have been isolated in about 105 CFU/mL from tracheal aspirates of rats from conventional animal facilities in Sao Paulo. The mycoplasma transmission by aerosol may happen from an infected rat to a healthy one at distances up to 120 cm. This condition also favors the technicians contamination. As this infection is unknown in humans, in this study the immunoblot profiles to M. pulmonis of sera from rats were compared to those presented by animal facility technicians. About 32 proteins from 11 to 230 kDa (kilodaltons) were recognized by the sera from rats naturally infected with M. pulmonis. Sera from technicians responsible for the cleaning and sanitation of cages of infected animals for more than seven years recognized about 10 proteins of this bacteria. Sera from individuals with shorter working time or that had never been exposed to such environment recognized few proteins. Proteins about 117 and 95 kDa were recognized by human and rat sera and by the negative controls. Although a positive human serum against M. pulmonis is unknown, this study established a temporary profile of protein recognition of human serum against such mycoplasma.