BACKGROUND: Cigarette smoking increases risk for multiple diseases. MicroRNAs regulate gene expression and may play a role in smoking-induced target organ damage. We sought to describe a microRNA signature of cigarette smoking and relate it to smoking-associated clinical phenotypes, gene expression, and lung inflammatory signaling. METHODS AND RESULTS: Expression profiling of 283 microRNAs was conducted on whole blood-derived RNA from 5023 Framingham Heart Study participants (54.0% women; mean age, 55±13 years) using TaqMan assays and high-throughput reverse transcription quantitative polymerase chain reaction. Associations of microRNA expression with smoking status and associations of smoking-related microRNAs with inflammatory biomarkers and pulmonary function were tested with linear mixed effects models. We identified a 6-microRNA signature of smoking. Five of the 6 smoking-related microRNAs were associated with serum levels of C-reactive protein or interleukin-6; miR-1180 was associated with pulmonary function measures at a marginally significant level. Bioinformatic evaluation of smoking-associated genes coexpressed with the microRNA signature of cigarette smoking revealed enrichment for immune-related pathways. Smoking-associated microRNAs altered expression of selected inflammatory mediators in cell culture gain-of-function assays. CONCLUSIONS: We characterized a novel microRNA signature of cigarette smoking. The top microRNAs were associated with systemic inflammatory markers and reduced pulmonary function, correlated with expression of genes involved in immune function, and were sufficient to modulate inflammatory signaling. Our results highlight smoking-associated microRNAs and are consistent with the hypothesis that smoking-associated microRNAs serve as mediators of smoking-induced inflammation and target organ damage. These findings call for further mechanistic studies to explore the diagnostic and therapeutic use of smoking-related microRNAs.