Prolactinomas are the most common pituitary tumors. The mechanisms of cell-cycle regulators underlying their invasive biological behavior and poor prognosis have not yet been fully clarified. We classified 48 human pro-lactinomas as invasive or non-invasive and determined cyclin D1, cyclin E1, p16, p27, Cdk2 and Cdk4 expression by immunohistochemistry analysis of tissue microarray constructs. Then we determined the diagnostic and prognostic value of the cell-cycle regulators expression in human prolactinomas. In this proof of principle study we found that nuclear p16 and p27 expression levels were much lower in invasive prolactinomas compared with non-invasive prolactinomas. Meanwhile, significantly higher cyclin D1 and cyclin E1 expression in invasive prolactinomas compared with normal pituitary or non-invasive prolactinomas. No difference was found in Cdk2 or Cdk4 protein levels in invasive or non-invasive prolactinomas. Regarding clinical outcome, the expression ratios of cyclin D1/p16 and cyclin E1/p27 were significantly positively correlated with clinically inferior outcome (P<0.001), while Cdk2 or Cdk4 expression showed no relationship with clinical outcome. Our findings indicate that the expression ratios of cyclin D1/p16 and cyclin E1/p27 are associated with invasion and clinic outcome of prolactinomas. We demonstrate the utility of combined histological analyses of prolactinomas for reliable prediction of tumor invasiveness and recurrence potential.