Background: The predictive value of thymidylate synthase (TYMS) to sensitivity to pemetrexed-based chemotherapy in advanced non-small cell lung cancer (NSCLC) patients is controversial. We conducted a meta-analysis of all relevant published data to assess the association of TYMS expression with the clinical outcomes of pemetrexed-based regimen in advanced NSCLC. Patients and Methods: We conducted an electronic search using using PubMed, Embase, OVID and Cochrane Library databases and manual search. Pooled odds ratio (OR) for the response rate and hazard ratio (HR) for the overall survival and progression free survival were calculated using the software Revman 5.0. Results: There were 11 studies (n=798) met our criteria for evaluation. Response rate to pemetrexed-based regimen was significantly higher in patients with low/negative TYMS (OR=2.96, 95% CI [1.81, 4.86] P<0.0001). Patients with low/negative TYMS who were treated with pemetrexed-based regimen had longer progression free survival (HR 0.50, 95% CI [0.41, 0.61] P < 0.00001) and overall survival (HR 0.41, 95% CI [0.22, 0.78] P=0.007) than those with high/positive TYMS. Conclusions: Low/negative TYMS expression was significantly associated with higher response rate, longer median survival and longer progression free survival for advanced NSCLC patients receiving pemtrexed-based chemotherapy. Hence, TYMS may be a potential predictor of sensitivity to pemtrexed-based chemotherapy in advanced NSCLC. Large scale prospective clinical trials are still warranted.