Objective: To explore the expressions and significances of CDK4 and Ki-67 in non-small cell lung cancer (NSCLC). Methods: We collected 271 cases of surgically resected NSCLC tissues and corresponding normal tissue, which admitted to Department of Thoracic Surgery. The expressions of CDK4 and Ki-67 were detected by immunohistochemistry. The correlation between CDK4 and Ki-67 expressions as well as the clinical significances were analyzed by statistical method. Results: Positive staining of CDK4 and Ki-67 were mainly in nucles. The positive expressions of CDK4 and Ki-67 in NSCLC, adenocarcinoma and squamous cell carcinoma were higher than those in the corresponding normal tissue (all P<0.001). The expressions of CDK4 and Ki-67 were no significant differences respectively between in the groups of adenocarcinoma and squamous cell lung cancer (all P>0.05), but there were significant differences in the groups of grades, clinical stages and lymph node metastasis or not of NSCLC (all P<0.01). The CDK4 expression showed positive correlation with Ki-67 expression in NSCLC, adenocarcinoma and squamous cell carcinoma respectively (r=0.520, 0.544, 0.501, all P<0.001). The patients with++/+++ expression CDK4 and Ki-67 have shorter survival time compared with those with −/+ expression, P=0.0019, P=0.0227 respectively. In univariate analysis, factors including CDK4 and Ki-67 expression, grade of differentiation, TNM stage and lymph node metastasis showed a significantly higher hazard ration for a poor prognosis (all P<0.01). Moreover, in multivariate analysis, the factors including TNM stage and CDK4 expression were independent prognostic factor (P=0.001, P=0.033). Conclusion: The higher expression of CDK4 may be correlate with the occurrence, development, and metastasis of NSCLC. Combined detection of CDK4 and Ki-67 proteins expressions may be valuable for early diagnosis and prognosis of NSCLC.