2-Acetyl-4-tetrahydroxybutylimidazole (THI), a by-product of Class Ⅲ caramel color, is generally recognized to cause lymphopenia in mammals. However, it remains unknown whether THI exposure during gestation and lactation causes damage to the immune system of offspring. In this study, pregnant Balb/c mice were gavaged with 0, 0.5, 2.5 and 12.5 mg/kg THI from gestation day (GD) 6 to postanal day (PND) 21, after which we treated another batch of dams from GD6 to PND21 and the offspring for 3 weeks after weaning with 0, 2, 10, 50 mg/L THI in drinking water respectively, and investigated the immunological anomalies of dams and offspring. The results showed that lymphopenia was observed in dams but not in weaning pups on PND21, which were exposed to THI during gestation and lactation. 2 mg/L THI and 2.5 mg/kg THI began to cause a remarkable reduction of the numbers of white blood cells and lymphocytes in dams. Besides both the cellular and the humoral immune response was not affected in weaning pups, which were measured by plaque-forming cell (PFC) assay and delayed-type hypersensitivity (DTH) assay respectively. Furthermore, THI could be detected in the plasma of dams with a dose-dependent manner, but not in that of both female and male weaning pups. In both male and female offspring being treated with 10 and 50 mg/L THI for another 3 weeks after weaning, lymphocytopenia was observed and T lymphocytes including CD4+ and CD8+ cells were significantly reduced in their spleens except lymph nodes. 10 and 50 mg/L THI treatment increased CD4+ and CD8+ single positive cells in thymus of female and male weaning mice. Mitogen-induced proliferation ability of T cells in the spleen and lymph nodes was impaired in female weaning mice exposed 50 mg/L THI, while male weaning mice treated with 10 and 50 mg/L THI showed impairment in the spleen but not lymph nodes. Based on the results in this study, no observed adverse effect level (NOAEL) for 3-week THI treatment in weaning mice was considered to be 2 mg/L (0.30 mg/kg bw for female mice and 0.34 mg/kg bw for male mice). And NOAEL for THI treatment in dams might be set to 0.5 mg/kg bw/day. Collectively from the perspective of NOAEL, offspring are not more sensitive than dams or adult mice.