Abstract The water channels aquaporins mediate a variety of immune functions. We previously demonstrated that aquaporin 4 (AQP4) is expressed by CD4 and CD8 T cells and blockade of AQP4 with small molecule inhibitor AER-270/271 significantly prolongs survival of heart allografts in two robust models of rejection. The goal of this study was to determine the effects of AQP4 blockade on T cells. Administration of AER-271 into naïve non-transplanted mice (250 μg i.p. every 6 h on d. 0–4) decreased numbers of circulating CD4 and CD8 T cells, but not B cells, by >90%, compared to untreated controls. The T cell frequencies and numbers in the secondary lymphoid organs (SLOs) were not significantly affected by AER-271, suggesting the lack of circulating T cells is not due to systemic depletion but rather to altered T cell trafficking. The effect of AER-271 treatment was transient, as circulating T cell numbers returned to pretreatment levels by d 21. AER-271 treated animals promptly rejected heart allografts transplanted 24 d after treatment cessation (MST 6 d, n=4). We next tested the effects of AQP4 blockade on Sphingosine-1 Phosphate Receptor 1 (S1PR1), a key mediator of T cell trafficking. S1PR1 mRNA expression was reduced in isolated mouse spleen T cells within 3 h of in vitro incubation with AER-270. Decreased S1PR1 mRNA expression translated into altered chemotaxis, as AER-270 reduced T cell migration toward S1PR1 ligand, Sphingosine-1 Phosphate (S1P), in a transwell system. Our data suggest that AER-270/271 treatment results in altered S1PR1 transcription thus changing T cell trafficking and prolonging allograft survival. Therefore, AQP4 blockade may be an attractive therapeutic strategy in transplantation and other immune-mediated diseases.