<i>Background:</i> Nitric oxide (NO) is involved in asthma pathogenesis and is synthesized by three isoforms of NO synthase, one of them encoded by <i>NOS1</i> gene. The CA-repeat and the C5266T SNP in <i>NOS1 </i>exon 29 have been associated with asthma and IgE levels. We thought to test the association of asthma and asthma-related phenotypes with the exon 29 CA-repeat and the C5266T SNP in a Colombian population sample. <i>Methods:</i> The CA-repeat and the C5266T SNP were genotyped in 167 asthmatics and 166 controls using PCR-based fragment length polymorphism and TaqMan assay. We also determined total and mite-specific IgE against <i>Blomia tropicalis </i>and <i>Dermatophagoides pteronyssinus</i>. <i>Results:</i> Three new CA-repeat alleles, 14, 23 and 24 repeats were detected. Allele comprising 16 repeats was associated with asthma (OR: 1.90 (CI 1.22–2.97, p<sub>c</sub> = 0.028) and low total (p<sub>c</sub> = 0.02) and specific IgE to <i>B. tropicalis</i> (p<sub>c</sub> < 0.0001) and <i>D. pteronyssinus</i> (p<sub>c</sub> < 0.0001). We found no association of the C5266T SNP and asthma or IgE levels. <i>Conclusion:</i><i>NOS1 </i>exon 29 CA-repeat may be a risk factor for asthma susceptibility and mite specific IgE response in a Colombian population.