Context: Exposure to ambient particulate air pollution is associated with increased cardiovascular and respiratory morbidity and mortality. It is necessary to understand causal pathways driving the observed health effects, particularly if they are differentially associated with particle size. Objectives: To investigate the effect of different size ranges of ambient particulate matter (PM) on gene and protein expression in an in vitro model. Materials and methods: Normal human tracheobronchial epithelium (NHTBE) three-dimensional cell constructs were exposed for 24 h to washed ambient PM of different sizes (size 1: 7–615 nm; size 2: 616 nm–2.39 µm; size 3: 2.4–10 µm) collected from a residential street. A human stress and toxicity PCR array was used to investigate gene expression and iTRAQ was used to perform quantitative proteomics. Results: Eighteen different genes of the 84 on the PCR array were significantly dysregulated. Treatment with size 2 PM resulted in the greatest number of genes with altered expression, followed by size 1 and lastly size 3. ITRAQ identified 317 proteins, revealing 20 that were differentially expressed. Enrichment for gene ontology classification revealed potential changes to various pathways. Discussion and conclusions: Different size fractions of ambient PM are associated with dysregulatory effects on the cellular proteome and on stress and toxicity genes of NHTBE cells. This approach not only provides an investigative tool to identify possible causal pathways but also permits the relationship between particle size and responses to be explored.