The hepatitis C virus nonstructural NS5A protein has roles in genome replication, virus assembly, and modulation of host pathways. NS5A is a phosphoprotein, and it has been proposed that differential phosphorylation could regulate the various roles of the protein. By SDS-PAGE, two forms of NS5A with distinct apparent molecular weights can be observed, referred to as basally phosphorylated and hyperphosphorylated species. However, the sites of phosphorylation on these two species have not been unequivocally identified, hampering our understanding of the function and regulation of NS5A. To address this, we purified tagged NS5A from cells harboring a replicating subgenomic replicon and analyzed the purified protein by mass spectrometry. We identified six peptide fragments containing 12 phosphorylated residues and were able to assign four of these to serines 146, 222, and 225 and threonine 348. A serine-rich peptide fragment spanning residues 221 to 240 was highly phosphorylated. Using mutagenesis, we identified roles for a subset of these phosphoacceptors in virus genome replication. We further showed that phosphorylation at S146 regulates hyperphosphorylation, and by generating a phospho-specific antibody targeted to pS222, we identified that S222 phosphorylation predominates in the hyperphosphorylated species. Last, by introducing phosphomimetic mutations across residues 221 to 240, we demonstrated changes in the mobility of the basally phosphorylated species suggestive of a sequential phosphorylation cascade within this serine-rich cluster. We propose that this regulation could drive a conformational switch between the dimeric structures of NS5A and could also explain the different functions of the protein in the virus life cycle.