Cladribine is a purine nucleoside analog developed to treat lymphoid malignancies. Reported therapeutic benefits for the autoimmune disease multiple sclerosis indicate additional immunomodulatory effects beyond the well-characterized cytotoxic activity causing lymphopenia. Here, we demonstrate that cladribine reduces the secretion of inflammatory cytokines and chemokines by murine and human dendritic cells, the most potent antigen-presenting cells. This compound also modulates the expression of the activation markers CD86 and MHC II. Furthermore, cladribine affects the T cell priming capacity of dendritic cells, resulting in reduced induction of interferon-γ- and tumor necrosis factor-α-producing T cells and increased induction of interleukin-10-producing T cells. These effects, observed at cladribine concentrations in the therapeutically relevant range of serum steady-state concentrations for leukemia and multiple sclerosis, confirm the immunomodulatory activity of cladribine.