Abstract Anti-CD3 antibody has been employed in several clinical settings. However, its effect on glucose metablolism has not been well investigated. In the current study, we investigated how anti-CD3 treatment affected blood glucose levels in mice. We found that anti-CD3 treatment induced immediate reduction of blood glucose as early as 3-4 hours post-treatment. Furthermore, we observed that a single dose of anti-CD3 treatment was able to correct hyperglycemia in all diabetic NOD mice. This glucose-lowering effect was not attributable to major T cell cytokines secreted by anti-CD3 activated T cells. Of interest, when tested in a normal strain of mice (C57BL/6), the C-peptide levels in anti-CD3-treated animals were significantly lower than those of control mice. Paradoxically, the anti-CD3-treated animals were highly tolerant to the exogenous glucose challenge. In addition, we found anti-CD3 treatment significantly induced activation of T and B cells in vitro and in vivo. Our further studies showed that anti-CD3 treatment lowered the glucose levels in T cell culture media, and increased the intracellular transportation of 2-NBDG particularly in activated T and B cells. In addition, injection of anti-CD3 antibodies upregulated Glut1 expression in spleen cells. The present study suggests that anti-CD3 therapy-induced hypoglycemia likely results from increased glucose transportation and consumption by the activated lymphocytes.