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Increase in air pollution caused by acidic gases, such as nitrogen dioxide, call for the need to study potential effects on the structure of textile materials. Wool is a natural protein fiber which is easily damaged due to rich reaction positions and cross-linked bonds in the fiber. This study reports, for the first time, effect of nitrogen dioxide, as a model acid gas pollutant, on the structure of wool. On the macro level, wool fabric undergoes strength loss after incubation in nitrogen dioxide for 6 h. Furthermore, although there was no obvious difference on the surface morphology as shown by SEM analysis, FTIR and Raman tests showed that the probable damage mechanism is by hydrolysis of amino bond, and oxidation of tyrosine residues and disulfide bonds, which was ascertained using XPS and amino acid analysis. In addition, crystallinity degree and α-helix structure content decreased after nitrogen dioxide treatment as shown by XRD and NMR results. These findings show that nitrogen dioxide has significant effect on the structure of wool. The study offers a novel approach for investigation of the interaction between gas pollutants and protein fiber, and provide information for protection of textiles against similar pollutants.