Methylenetetrahydrofolate reductase (MTHFR) enzyme is essential for transmethylation reactions including DNA methylation and DNA synthesis and thereby may contribute to cancer prognosis. In our study, a total of 1,004 advanced non-small cell lung cancer (NSCLC) patients receiving first-line, platinum-based chemotherapy regimens were used for genotyping 10 tag single nucleotide polymorphisms (SNPs) of MTHFR. Association was assessed between the SNPs and treatment outcomes. We found that polymorphism of rs1537514 showed the most significant effect: heterozygote associated with better clinical benefit (P = 0.002) and decreased risk of grade 3 or 4 gastrointestinal toxicity (P = 0.027), while the mutant homozygote associated with increased risk of severe gastrointestinal toxicity (P = 0.031) and thrombocytopenia (P = 009). The heterozygotes of exon polymorphisms (rs1801131, rs1801133) also yielded better clinical benefit (P = 0.030 for rs1801131) and decreased risk of severe gastrointestinal toxicity (P = 0.004 for rs1801131) or thrombocytopenia (P = 0.016 for 1801133). However, overall survival (OS) and progression-free survival (PFS) did not differ for the MTHFR polymorphisms, except for heterozygote of rs1537514 showing significant effects with better PFS (P = 0.022). Clinical factors as age, gender, and smoking status had significant effects for the OS (P = 0.003, 0.002, and 0.012, respectively) while performance status and chemotherapy regimens for PFS (P = 0.001 and 3.9 × 10(-6), respectively). The results indicate that a heterozygous advantage may exist in certain MTHFR variants, and the polymorphisms (especially rs1537514) may play a predictive role of treatment efficacy and adverse effects in NSCLC patients treated with platinum-based chemotherapy.