Rfa2 is a ssDNA (single-stranded DNA)-binding protein that plays an important role in DNA replication, recombination and repair. Rfa2 is regulated by phosphorylation, which alters its protein-protein interaction and protein-DNA interaction. In the present study, we found that the Pph3-Psy2 phosphatase complex is responsible for Rfa2 dephosphorylation both during normal G -phase and under DNA replication stress in Candida albicans. Phosphorylated Rfa2 extracted from pph3 Δ or psy2 Δ G cells exhibited diminished binding affinity to dsDNA (double-stranded DNA) but not to ssDNA. We also discovered that Cdc28 (cell division cycle 28) and Mec1 are responsible for Rfa2 phosphorylation in G-phase and under DNA replication stress respectively. Moreover, MS revealed that the domain of Rfa2 that was phosphorylated in G-phase differed from that phosphorylated under the stress conditions. The results of the present study imply that differential phosphorylation plays a crucial role in RPA (replication protein A) regulation.