Excess production of nitric oxide and reactive nitrogen intermediates causes nitrosative stress on cells. Schizosaccharomyces pombe was used as a model to study the cell cycle regulation under nitrosative stress response. We discovered a novel intra-S-phase checkpoint that is activated in S. pombe under nitrosative stress. The mechanism for this intra-S-phase checkpoint activation is distinctly different than previously reported for genotoxic stress in S. pombe by methyl methane sulfonate. Our flow cytometry data established the fact that Wee1 phosphorylates Cdc2 Tyr15 which leads to replication slowdown in the fission yeast under nitrosative stress. We checked the roles of Rad3, Rad17, Rad26, Swi1, Swi3, Cds1, and Chk1 under nitrosative stress but those were not involved in the activation of the DNA replication checkpoint. Rad24 was found to be involved in intra-S-phase checkpoint activation in S. pombe under nitrosative stress but that was independent of Cdc25. 