CD103 plays an important role in the destruction of islet allografts, and previous studies found that a CD103 immunotoxin (M290-Saporin, or M290-SAP) promoted the long-term survival of pancreatic islet allografts. However, systemic toxicity to the host and the bystander effects of M290-SAP obscure the underlying mechanisms of action and restrict its clinical applications. To overcome these shortcomings, anti-CD103 M290 was conjugated to different cytotoxic agents through cleavable or uncleavable linkages to form three distinct antibody–drug conjugates (ADCs): M290-MC-vc-PAB-MMAE, M290-MC-MMAF, and M290-MCC-DM1. The drug-to-antibody ratio (DAR) and the purity of the ADCs were determined by HIC–HPLC and SEC–HPLC, respectively. The binding characteristics, internalization and cytotoxicity of M290 and the corresponding ADCs were evaluated in vitro. The cell depletion efficacies of the various M290–ADCs against CD103-positive cells were then evaluated in vivo. The M290–ADCs maintained the initial binding affinity for the CD103-positive cell surface antigen and then quickly internalized the CD103-positive cell. Surprisingly, all M290–ADCs potently depleted CD103-positive cells in vivo, with high specificity and reduced toxicity. Our findings show that M290–ADCs have potent and selective depletion effects on CD103-expressing cells in immunocompetent mice. These data indicate that M290–ADCs could potentially serve as a therapeutic intervention to block the CD103/E-cadherin pathway.