The Ser/Arg-rich (SR) proteins constitute a family of highly conserved nuclear phosphoproteins that are involved in many steps of mRNA metabolism. Previously, we demonstrated that shuttling SR proteins can associate with translating ribosomes and enhance translation of reporter mRNAs both in vivo and in vitro. Here, we show that endogenous, cytoplasmic splicing factor 2/alternative splicing factor (SF2/ASF) associated with the translation machinery is hypophosphorylated, suggesting that the phosphorylation state of the Arg-Ser-rich (RS) domain may influence the role of SF2/ASF in cytoplasmic RNA processing. In agreement, we show that mutations mimicking a hypophosphorylated RS domain strongly increased SF2/ASF binding to cytoplasmic mRNA and its activity in translation. We also demonstrate that, whereas the RS domain is not required for the function of SF2/ASF in mRNA translation in vivo or in vitro, its second RNA recognition motif (RRM)2 plays a critical role in this process. Taken together, these data suggest that RS-domain phosphorylation may influence the association of SF2/ASF with mRNA, whereas RRM2 may play an important role in mediating protein-protein interactions during translation. These data are consistent with a model whereby reversible protein phosphorylation differentially regulates the subcellular localization and activity of shuttling SR proteins.