PCTAIRE kinase 3 (PCTK3)/cyclin dependent kinase 18 (CDK18) is a member of the CDK family. Although the PCTK3 is relatively more abundant in post‐mitotic cells, their enzymatic characteristic and physiological function have remained unknown because of unidentified its activator(s). Here, we describe the mechanisms of catalytic activation of PCTK3 by cyclin A2 and cAMP‐dependent protein kinase (PKA). Using a pull‐down experiment with HEK293T cells, cyclin A2 and cyclin E1 were identified as proteins that interacted with PCTK3. An in vitro kinase assay using retinoblastoma protein as the substrate showed that PCTK3 was specifically activated by cyclin A2 but not by cyclin E1. Furthermore, immunocytochemistry analysis showed that PCTK3 colocalized with cyclin A2 in the cytoplasm and regulated cyclin A2 stability. Amino acid sequence analysis revealed that PCTK3 contained four putative PKA phosphorylation sites. In vitro and in vivo kinase assays showed that PCTK3 was phosphorylated by PKA at Ser12, Ser66, and Ser109, and that PCTK3 activity significantly increased via phosphorylation at Ser12by PKA, even in the absence of cyclin A2. In the presence of cyclin A2, PCTK3 activity was comparable to CDK2 activity. We also found that PCTK3 knockdown in HEK293T cells induced morphological changes and polymerized actin accumulation in peripheral areas. In addition, we observed that cofilin phosphorylation and cell motility in PCTK3 knockdown HEK293T cells was markedly increased as compared with that in control cells. Taken together, our results provide the first evidence for the mechanisms of catalytic activation of PCTK3 by cyclin A2 and PKA, and a physiological function of PCTK3.