Neutrophil elastase (NE) is a powerful protease capable of degrading various protein components of the extracellular matrix, coagulation and component cascades. Local production of NE is involved in invasion and associated with a poor lung cancer prognosis. We propose that NE is important in lung cancer development, especially in the invasive phase. We tested this hypothesis using 344 cases and 299 controls. We detected single-nucleotide polymorphisms at the NE locus by denaturing high-performance liquid chromatography analysis, DNA sequencing and cloning of the NE gene promoter region. We performed transfection analysis, using relative luciferase activity on a human lung cancer cell line, to determine the regulatory role of the detected single-nucleotide polymorphisms in setting transcription levels of NE promoter. Two new single-nucleotide polymorphism markers, REP_A (T and G) and REP_B2 (G and A), were detected. There was no GG at REP_A and very little AA at REP_B2. TT and TG were contrasting allele types at REP_A, and GG and AA/AG were contrasting allele types at REP_B2. Measured by odds ratio, the TT type at REP_A or GG type at REP_B2 was associated with a 2.3 or 1.4 times higher risk of developing lung cancer than the TG and AA/AG types, respectively. When assessing the combined effects of the high-risk alleles, the odds ratio was 24.8. We demonstrated a 1.9-fold increase of relative luciferase activity in the T-G construct compared with the G-A construct, providing evidence that the TT-GG type correlates with a high NE level. Our findings indicate an important role for NE in lung cancer development.