Cyclin Y-like 1 (Ccnyl1) is a newly identified member of the cyclins family, which was similar to Cyclin Y.However, the function of Ccnyl1 is poorly characterized.Here we found Ccnyl1 was most abundantly expressed in the testis of mice, which was about seven times higher than the level of Cyclin Y.Consistently, male Ccnyl1-/-mice were infertile, whereas both male and female Cyclin Y-/-mice displayed normal fertility.These results suggested that Ccnyl1, but not Cyclin Y, was indispensable for male fertility.The sperm obtained from Ccnyl1-/-mice displayed significantly impaired motility, and represented with thinning and/or bent of the annulus and/or a bent of head.We found that the protein level, but not the mRNA, of Cdk16 was significantly decreased in testis of Ccnyl1-/-mice.Further study demonstrated that Ccnyl1 interacted with Cdk16, and the interaction between Ccnyl1 and Cdk16 mutually increased the stabilities of the two protein.Moreover, the interactions between Ccnyl1 and Cdk16 also robustly increased the kinase activity of Cdk16.In addition, we fully identified the phosphorylation sites of Cdk16 by mass spectrometry, and revealed that several phosphorylation modifications on the N-terminal region of Cdk16 were indispensable for the Ccnyl1 binding and the modulation of Cdk16 kinase activity.Our results therefore reveal a previously unrecognized role of Ccnyl1 in regulating the spermatogenesis through the interaction and modulation of Cdk16.