Pituitary adenomas constitute 15-20% of intracranial neoplasms. Previously we reported that cyclin-dependent kinase 5 (CDK5) is upregulated in pituitary tumors associated with activating protein p35, and plays an essential role in pituitary adenomas progression. Here we explored the mechanisms of CDK5 signaling in prolactin pituitary adenomas. Our data indicate that p35 expression and CDK5 activity are both significantly increased in human invasive prolactin pituitary adenomas as compared to noninvasive forms of pituitary adenomas. Inhibition of CDK5 activity suppressed cell migration and invasive ability in GH3 rat pituitary cells. We identified that CDK5 phosphorylates serine 229 residue (Ser-229) of kinase insert domain receptor (KDR), also known as VEGFR-2, in prolactin pituitary adenomas. Phosphorylation of Ser-229 is required for proper KDR surface localization. Phosphorylated Ser-229 in KDR (pSer-229) levels are significantly higher in noninvasive and invasive prolactin pituitary adenomas compared to normal pituitary tissues. In addition, our data indicated that higher KDR pSer-229 correlates with worse prognosis in patients with prolactin pituitary adenomas. In summary, our results illustrated that CDK5-mediated KDR phosphorylation controls prolactin pituitary adenoma progression and KDR pSer-229 serves as a potential prognostic biomarker for both noninvasive and invasive pituitary adenomas.