Macrophages as an early stage of immune responses form a bridge between innate and acquired immunity and shape the adaptive immune response. The immunoregulatory functions of macrophages in hosts with a prolonged exposure to a diabetic milieu remain to be determined. The levels, phenotype, and immunity including antigen-presenting ability, phagocytosis and immunogenicity of F4/80⁺ splenic macrophages (SPMs), and peritoneal exudates macrophages (PEMs) were detected in age-matched control mice and mice with streptozotocin (STZ)-induced diabetes for 16 weeks. The numbers of F4/80⁺ SPMs and PEMs significantly decreased in STZ-induced diabetic mice, compared with age-matched non-diabetic mice (control) at 16 weeks after diabetes induction. Functional analysis showed that F4/80⁺ SPMs and PEMs in STZ-induced diabetic mice exhibit significantly lower immunogenicity and nonopsonic phagocytosis to allogeneic T cells than those of control mice both in vitro and in vivo. Coincidently, the antigen-presenting capacity of F4/80⁺ PEMs, but not F4/80⁺ SPMs, in mice with STZ-induced diabetes for 16 or more weeks is also significantly lower than that of control mice. Our results showed that total cell number and immune function of F4/80⁺ macrophages were significantly defective in mice with a prolonged exposure to a diabetic milieu, which may be a mechanism responsible for the increased macrophage-related complications in diabetic patients such as the high prevalence of infection and cardiovascular mortality.