Checkpoint kinase 1 (CHK1), a Ser/Thr protein kinase, is modified by the K63-linked ubiquitin chain in response to genotoxic stress, which promotes its nuclear localization, chromatin association, and activation. Interestingly, this bulky modification is linked to a critical residue, K132, at the kinase active site. It is unclear how this modification affects the kinase activity and how it is removed to enable the release of CHK1 from chromatin. Herein, we show that the K63-linked ubiquitin chain at CHK1’s K132 residue has an inhibitory effect on the kinase activity. Furthermore, we demonstrate that this modification can be removed by ubiquitin-specific protease 3 (USP3), a deubiquitinating enzyme that targets K63-linked ubiquitin chains. Wild-type USP3, but not the catalytically defective or nuclear localization sequence-deficient mutants, reduced CHK1 K63-linked ubiquitination. Conversely, USP3 knockdown elevated K63-linked ubiquitination of the kinase, leading to prolonged CHK1 chromatin association and phosphorylation. Paradoxically, by removing the bulky ubiquitin chain at the active site, USP3 also increased the accessibility of CHK1 to its substrates. Thus, our findings on the dual roles of USP3 (namely, one to release CHK1 from the chromatin and the other to open up the active site) provide further insights into the regulation of CHK1 following DNA damage.