Background In the present study, the researchers evaluated the presence of human anti-mouse antibodies (HAMA) in a normal population and laboratory animal care providers for the first time in the world. Also, the cause of HAMA incidence in the human body through a close contact with mice was identified. Methods The study population consisted of 40 laboratory animal care providers aged between 24 and 57 years with a close contact with mice (e.g. taking care of mice, feeding mice, etc.) and 40 individuals of the same age as the above group with no contact with mice. HAMA was measured in both the case and control groups using sandwich enzyme-linked immunosorbent assay (ELISA) method. Data were analyzed using SPSS 18. Univariate and multivariate linear regression and independent t-test were used. The significance of results was measured based on p < 0.05. Results The present study revealed that the animal care providers had (p = 000) a higher titer of HAMA (4.95 ng/mL) in their blood than the control group (1.67 ng/mL). Also, the individuals in the case group (exposed to mice) were more allergic (43.6%) than those in the control group (15%) (p = 0.003). Conclusions The results of this study revealed that exposure to mice in laboratory care centers can cause production of HAMA in the human body but its titer is possibly lower in Iranian working staff than those in the other parts of the world.