Cell cycle control can prevent excessive proliferative response in the pituitary homeostasis. Cyclin dependent kinases (Cdks) are modulated by cyclins or Cdk inhibitors, such as p21 and p27, which can regulate cell cycle progression from the G1 to S phases. This study was conducted to evaluate the levels and the promoter region methylation status of p21 and p27 in prolactinomas (PRL) and analyze their association with clinicopathologic features. We found high-p21 level cases were featured by 5/23 and H-scores 142.3 +/- 23.7 in invasive-PRL specimens, and 19/25 and 221.3 +/- 45.4 in non-invasive specimens (x(2) = 14.11, p = 0.000), while high-p27 level cases were featured by 6/23 and H-scores 129.8 +/- 31.1 in invasive-PRL specimens, and 18/25 and 197.1 +/- 46.6 in non-invasive specimens (x(2) = 10.11, p = 0.001). A similar trend was also observed for p21 and p27 protein levels in PRL specimens through western-blot (P < 0.01, respectively). The Ki-67 index was much higher in invasive specimens than in non-invasive specimens (x(2) = 10.10, p = 0.001). Average 33 CpG sites per sample were analyzed by using MALDI-TOF Mass array, and 7/33 CpG sites methylation levels of p27 were higher than 50%. There existed significant differences in 4 CpG sites between invasive specimens and non-invasive specimens (p < 0.01). We found that D2 receptor was closely correlated with p21 levels (P < 0.05, r = 0.567) and p27 levels (P < 0.05, r = 0.591). In PRL, the deficiency in p21 and p27 contributed to the tumor proliferation and migration and Cdk inhibitors may be used as a new therapeutic approach.