The mouse anti-human CD3 monoclonal antibody such as OKT3 is a potent immunosuppressive agent used in clinical transplantation to manipulate T-cell functions and prevent acute allograft rejection. However, the broad use of anti-CD3 antibody in clinical treatment was severely limited by the side effects of human anti-mouse antibody response and cytokine release syndrome. In this study, on the basis of a murine anti-human CD3 antibody yCD3 obtained in our previous work, a novel engineered anti-human CD3 antibody fragment (i.e. V(H)-Linker-V(L)-Hinge-CH(3)) was constructed with computer-guided molecular design method to avoid the clinical side effects. According to the distance geometry and intra-molecular interaction, the hinge region was re-designed and different from the parental hinge region in human IgG1. With the novel hinge region, the cysteine residues in hinge were exposure and prone to form the disulfide bond. Therefore, a novel bivalent antibody fragment named as mini-yCD3 was obtained. Mini-yCD3 displayed similar antigen-binding affinity and specificity to yCD3. Importantly, mini-yCD3 was shown to be much less potent in the induction of T-cell proliferation, cytokine release (interferon-gamma and interleukin-2) and early activation marker expression on the cell surface (CD69 and CD25) than parental yCD3. Furthermore, mini-yCD3 was effective in modulating T-cell receptor/CD3 and inhibiting mixed lymphocyte reaction with similarity as yCD3. In conclusion, the constructed mini-yCD3 was much less mitogenic to T cells but retained potent immunosuppression, suggesting it might be an alternative to yCD3 as an immunosuppressive drug with less immunogenicity and toxicity for clinical application.