Abstract: Cyclin dependent kinase (Cdk) governs cell cycle progression and its kinase activity fluctuates during cell cycle. Mitotic exit pathways are responsible for the inactivation of Cdk after chromosome segregation by promoting the release of a nucleolar sequestered phosphatase Cdc14, which antagonizes Cdk. In the budding yeast Saccharomyces cerevisiae, mitotic exit is controlled by FEAR (Cdc fourteen early anaphase release) and MEN (mitotic exit network) pathways. In response to DNA damage, two branches of the DNA damage checkpoint, Chk1 and Rad53, are activated in budding yeast to prevent anaphase entry and mitotic exit, allowing cells more time to repair damaged DNA. Here we present evidence indicating that yeast cells negatively regulate mitotic exit through two distinct pathways in response to DNA damage. Rad53 prevents mitotic exit by inhibiting MEN pathway, whereas Chk1 pathway prevents FEAR pathway dependent Cdc14 release in the presence of DNA damage. In contrary to previous data, Rad53 pathway negatively regulates MEN independently of Cdc5, a polo-like kinase essential for mitotic exit. Instead, defective Rad53 pathway alleviates the inhibition of MEN by Bfa1.