Background: Nitric oxide (NO) is involved in asthma pathogenesis and is synthesized by three isoforms of NO synthase, one of them encoded by NOS1 gene. The CA-repeat and the C5266T SNP in NOS1 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. Methods: 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 Blomia tropicalis and Dermatophagoides pteronyssinus. Results: 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, pc = 0.028) and low total (pc = 0.02) and specific IgE to B. tropicalis (pc < 0.0001) and D. pteronyssinus (pc < 0.0001). We found no association of the C5266T SNP and asthma or IgE levels. Conclusion:NOS1 exon 29 CA-repeat may be a risk factor for asthma susceptibility and mite specific IgE response in a Colombian population.