Rationale: Air pollution may contribute to the development of allergic diseases, including asthma, by enhancing immune responses to allergen, but the effects on microRNA, messenger RNA, and inflammatory markers remain unexplored. Objectives: To determine if acute exposure to diesel exhaust and/or allergen alters expression of microRNA and genes with effects on inflammatory markers associated with asthma. Methods: Fifteen atopic participants were exposed in a crossover trial to inhaled filtered air or diesel exhaust (DE; 300 μg particulate matter with aerodynamic diameter less than or equal to 2.5 μm/m3), followed by saline-controlled, segmental allergen challenge. Lung inflammatory markers, gene expression, and microRNAs were measured in bronchial brushings, bronchial wash, or bronchoalveolar lavage 48 hours after exposure. Results: DE + saline and DE + allergen significantly modulated the highest number of microRNAs and messenger RNAs. Allergen exposure significantly modulated microRNAs, including miR-324-5p, miR-132-3p, and miR-183-5p, and genes, including cyclin-dependent kinase inhibitor 1C (CDKN1A) and nuclear factor κB inhibitor α (NFKBIA), but DE did not significantly modify this effect. Inflammatory markers, including bronchoalveolar lavage eosinophil cell percentages and eosinophil cationic protein, were significantly increased, whereas bronchoalveolar lavage epithelial cell percentages were reduced after allergen exposure. Conclusions: Expression of genes and microRNAs associated with bronchial immune responses were significantly modulated by allergen and DE. Clinical trial registered with www.clinicaltrials.gov (NCT01699204).