While several protein serine/threonine kinases control cytokine production by T cells, the roles of serine/threonine phosphatases are largely unexplored. Here, we analyzed the involvement of protein phosphatase 1α (PP1α) in cytokine synthesis following costimulation of primary human T cells. Small interfering RNA (siRNA)-mediated knockdown of PP1α (PP1KD) or expression of a dominant negative PP1α (D95N-PP1) drastically diminished interleukin-10 (IL-10) production. Focusing on a key transcriptional activator of human IL-10, we demonstrate that nuclear translocation of NF-B was significantly inhibited in PP1KD or D95N-PP1 cells. Interestingly, knockdown of cofilin, a known substrate of PP1 containing a nuclear localization signal, also prevented nuclear accumulation of NF-B. Expression of a constitutively active nonphosphorylatable S3A-cofilin in D95N-PP1 cells restored nuclear translocation of NF-B and IL-10 expression. Subpopulation analysis revealed that defective nuclear translocation of NF-B was most prominent in CD4+ CD45RA– CXCR3– T cells that included IL-10-producing TH2 cells. Together these findings reveal novel functions for PP1α and its substrate cofilin in T cells namely the regulation of the nuclear translocation of NF-B and promotion of IL-10 production. These data suggest that stimulation of PP1α could limit the overwhelming immune responses seen in chronic inflammatory diseases.