Mechanisms used by positive-stranded RNA viruses to control the translation-replication switch remain largely unexplored. Using metabolic labeling coupled with quantitative proteomics analyses, we unraveled the protein composition of temporal ribonucleoprotein complexes (RNPs) isolated during early and late enterovirus A71 (EV-A71) infection. Comparing the RNP components at the eclipse and maturation phase, representing early and late infection, revealed RNP remodeling over time by exchanging nuclear with cytoplasmic proteins. Among the nuclear proteins, EV-A71 infection induced the phosphorylation and cytoplasmic re-localization of nuclear SR proteins. During early infection, phosphorylated SR proteins cofractionated with the translation machinery rather than the replication organelles. During late infection, this co-localization, as well as the EV-A71-induced phosphorylated SR proteins, was no longer detected. Inhibition of SR protein phosphorylation by the kinase inhibitors SRPKIN-1 and TG003 significantly reduced the replication of several enteroviruses. Our results demonstrate the importance of phosphoregulation of SR proteins during enterovirus replication and reveal a potential target for broad-spectrum antivirals.