Activation of T cell factor-4 (TCF-4) is causally linked to the development of lung carcinoma, while the mechanism of sequence-dependent TCF-4 activity is still obscure. Using reverse transcription-polymerase chain reaction (RT-PCR), here, we demonstrated that sequences of exon 11 in TCF-4 were present in lung carcinoma cells but not in normal lung epithelial cells. Loss of exon 11 in TCF-4 inhibited TCF-4-induced cell growth of lung carcinoma and prolonged the survival time of Lewis lung carcinoma (LLC) tumor-bearing mice. Mechanistically, loss of exon 11 in TCF-4 attenuated the binding activity between TCF-4 protein and its canonical binding site, inhibited TOP/FOP luciferase activity and suppressed mRNA expression of Wnt signaling targets. By performing truncated and site-directed mutations, we further demonstrated that the 16th amino acid serine in exon 11 was responsible for TCF-4-mediated Wnt signaling. In vivo experiments indicated that a mutation of the 16th amino acid serine in exon 11 of TCF-4 could mimic the anti-tumor effect of Wnt signaling inhibitor. Taken together, we identified a serine determining the transcriptional activity of TCF-4 in lung carcinoma cells, and sequencing of TCF-4 mRNA might be an effective strategy to evaluate the Wnt pathway activation and prognosis in lung cancer.