The circadian oscillator is composed of a transcription/translation-based autoregulatory feedback loop in which Cryptochromes and Periods function as negative regulators for their own gene expression. Although post-translational modifications such as phosphorylation of these regulators appear crucial for circadian time-keeping mechanism, less is known about responsible protein kinases and their contribution to the function of the regulators. We found that mitogen-activated protein kinase (MAPK) associates with and phosphorylates mouse Cryptochromes (mCRY1 and mCRY2). Mass spectrometry analysis identified Ser265 and Ser557 of mCRY2 to be in vitro phospho-acceptor residues. Mutations of both the Ser residues to Ala completely abolished MAPK-mediated mCRY2 phosphorylation, suggesting that the two residues are the principal phosphorylation sites in mCRY2. Similarly, MAPK phosphorylates mCRY1 at Ser247, a site corresponding to Ser265 of mCRY2. An effect of the Ser phosphorylation was investigated by mutating Ser247 of mCRY1 and Ser265 of mCRY2 to Asp, which resulted in attenuation of each mCRYs' ability to inhibit BMAL1: CLOCK-mediated transcription, whereas a similar mutation at Ser557 of mCRY2 induced no measurable change in its activity. These results illustrate a model of MAPK-mediated negative regulation of mCRY function by phosphorylation at the specific Ser residue.