Alemtuzumab (anti-CD52 mAb) provides long-lasting disease activity suppression in relapsing-remitting multiple sclerosis (RRMS). The objective of this study was to characterize the immunological reconstitution of T cell subsets and its contribution to the prolonged RRMS suppression following alemtuzumab-induced lymphocyte depletion. The study was performed on blood samples from RRMS patients enrolled in the CARE-MS II clinical trial, which was recently completed and led to the submission of alemtuzumab for U.S. Food and Drug Administration approval as a treatment for RRMS. Alemtuzumab-treated patients exhibited a nearly complete depletion of circulating CD4(+) lymphocytes at day 7. During the immunological reconstitution, CD4(+)CD25(+)CD127(low) regulatory T cells preferentially expanded within the CD4(+) lymphocytes, reaching their peak expansion at month 1. The increase in the percentage of TGF-β1-, IL-10-, and IL-4-producing CD4(+) cells reached a maximum at month 3, whereas a significant decrease in the percentages of Th1 and Th17 cells was detected at months 12 and 24 in comparison with the baseline. A gradual increase in serum IL-7 and IL-4 and a decrease in IL-17A, IL-17F, IL-21, IL-22, and IFN-γ levels were detected following treatment. In vitro studies have demonstrated that IL-7 induced an expansion of CD4(+)CD25(+)CD127(low) regulatory T cells and a decrease in the percentages of Th17 and Th1 cells. In conclusion, our results indicate that differential reconstitution of T cell subsets and selectively delayed CD4(+) T cell repopulation following alemtuzumab-induced lymphopenia may contribute to its long-lasting suppression of disease activity.