The aim of this study was to investigate the efficacy of 11C-choline PET/CT imaging for lung cancer and the correlation between choline uptake of lung cancer tissue and the expression of choline kinase (ChoK), phosphorylcholine-cytidyl transferase and Ki-67 index. Between March 2008 and June 2010, 53 patients diagnosed or suspected of having lung cancer underwent integrated 11C-choline PET/CT and contrast-enhanced CT scans before surgery. After surgery, specimens from 42 patients diagnosed with lung cancer were used to detect the expression of ChoK, phosphorylcholine-cytidyl transferase and the Ki-67 index. The PET/CT results were analyzed using visual methods and the standardized uptake value (SUV) of lesions was measured using semi-quantitative methods. Finally, the analyzed results were compared to the histopathological results. The accuracy of the 11C-choline PET/CT for diagnosing lung cancer was 81.13% (43/53), compared with 71.70% (38/53) for CT scanning. The difference was not statistically significant (P=0.61). The accuracy of 11C-choline PET/CT for diagnosing lymph nodes was 83.76% (227/271), compared with 66.79% (181/271) for CT scanning. This difference was statistically significant (P=0.04); the SUVmean value of lesions correlated positively with the Ki-67 index (r=0.51, p=0.002). Of the 35 patients with positive 11C-choline PET results, 29 (82.86%) overexpressed ChoK, 26 (74.29%) overexpressed phosphorylcholine-cytidyl transferase. The seven patients with negative 11C-choline PET results did not exhibit overexpression of ChoK or phosphorylcholine-cytidyl transferase; the SUVmean value correlated positively with the expression of both ChoK and phosphorylcholine-cytidyl transferase (r=0.52, p=0.001; r=0.37, p=0.029). In conclusion, compared with contrast-enhanced CT, 11C-choline PET offers nodal staging with higher accuracy. The SUV value of PET is correlated with the proliferation of tumor cells and the mechanism of PET imaging is associated with the overexpression of ChoK and phosphorylcholine-cytidyl transferase.