The long-lasting clinical response by lymphoma patients to anti-CD20 therapy has been attributed to the induction of an anti-tumor adaptive immunity. We previously demonstrated that a CD4-dependent mechanism is responsible for the long-term protection of CD20+ tumor-bearing mice by anti-CD20 treatment. Here, we compare tumor immunity in tumor-bearing animals that did or did not receive anti-CD20 treatment. Splenic CD4+ FoxP3+ regulatory T cells (Tregs) expanded substantially in untreated mice that exhibited then a reduced survival, whereas Tregs depletion led to long-term survival of the animals, suggesting the establishment of a Treg-dependent immunosuppressive environment after tumor injection. Strikingly, anti-CD20 therapy reversed the initial expansion of Tregs, and was accompanied by a marked increase in the number of Th1 cells, with no detectable change in Th2 and Th17 cell numbers. Interleukin-12 serum level was also increased by the anti-CD20 treatment, and activated myeloid dendritic cells producing interleukin-12 could be detected in lymph nodes of treated animals, while interferon-γ blockade strongly reduced survival. Also, CD4+ effector memory T cells were evidenced in surviving animals, and the transfer of CD4+ T cells induced long-term protection. Thus, anti-CD20 therapy promotes strong anti-tumor adaptive immunity, opposes Treg expansion and inhibits tumor cells from maintaining an immunosuppressive environment.