Cell proliferation and cellular quiescence/G 0  phase must be regulated in response to intra-/extracellular environments, and such regulation is achieved by the orchestration of protein kinases and protein phosphatases. Here, we investigated fission yeast potential orthologs (Cek1, Ppk18 and Ppk31) of the metazoan Greatwall kinase (Gwl), which inhibits type-2A protein phosphatase with B55 subunit (PP2A B55  ) by phosphorylating and activating the PP2A B55  inhibitors, α-endosulfine/ARPP-19 (Ensa/ARPP-19). Gwl and Ensa/ARPP-19 regulate mitosis; however, we found Ppk18, Cek1 and Mug134/Igo1, the counterpart of Ensa/ARPP-19, are not essential for normal mitosis but regulate nitrogen starvation (-N)-induced proper G 0  entry and maintenance. Genetic and biochemical analyses indicated that the conserved Gwl site (serine 64) was phosphorylated in the G 0  phase in a Ppk18-dependent manner, and the phosphorylated Mug134/Igo1 inhibited PP2A B55  in vitro. The alanine substitution of the serine 64 caused defects in G 0  entry and maintenance as well as the mug134/igo1 +  deletion. These results indicate that PP2A B55  activity must be regulated properly to establish the G 0  phase. Consistently, simultaneous deletion of the B55 gene with mug134/igo1 +  partially rescued the Mug134/Igo1 mutant phenotype. We suggest that in fission yeast, PP2A B55  regulation by the Ppk18-Mug134/Igo1 pathway is required for G 0  entry and establishment of robust viability during the G 0  phase.