Cyclophosphamide (CTX), a commonly used chemotherapeutic agent can enhance immune responses. The ability of CTX to promote the proliferation of effector T cells and abrogate the function of regulatory T cells (Tregs) has been described. In this study, we examined the effects of CTX treatment on dendritic cell (DC) subsets and the subsequent outcome on the effector and suppressive arms of adaptive immunity. In secondary lymphoid tissues, tissue-derived migratory DCs (migratory DCs), lymphoid tissue-resident DCs (resident DCs), and plasmacytoid DCs (pDCs) are well described. CTX has profound and selective cytotoxic effects on CD8(+) resident DCs, but not skin-derived migratory DCs or pDCs in lymph nodes (LNs) and spleen, causing an imbalance among these DC subsets. CTX treatment increases the potency of DCs in antigen presentation and cytokine secretion, and partially inhibits the suppressor activity of Tregs. Adoptive transfer of CD8(+) DCs can reconstitute this population in regional draining LNs and abrogate the immune-enhancing effects of CTX in vivo. These findings demonstrate that CTX may improve immune responses by preferentially depleting CD8(+) lymphoid-resident DCs, which leads to diminished Treg suppression and enhanced effector T-cell function in vivo.