Although more than half of genomic loci are believed to have antisense transcription, whether antisense transcription is involved in cytokine expression has not been studied. In this study, we show that some loci of innate immunity related genes do have antisense transcripts. We investigated the effect of several antisense RNAs, including anti-4-1BBL, anti-p100, and anti-IL-1β, on their cognate sense gene's expression in macrophages. We found that overexpression of antisense IL-1β transcript suppressed IL-1β expression. Anti-IL-1β is complementary to the sequence in the 5' upstream region of the IL-1β promoter. Its mediated inhibition of IL-1β production occurred at the transcriptional level. Anti-IL-1β did not alter the methylation status of the IL-1β promoter. However, chromatin immunoprecipitation assays revealed that the anti-IL-1β transcript can change the chromatin structure of the IL-1β promoter by decreasing H3K4 trimethylation on the promoter, which is at least part of the mechanism underlying the reduced binding of RNA polymerase II to the IL-1β promoter upon anti-IL-1β expression. Our data suggest that some antisense transcripts of innate immunity-related genes play a role by regulating cytokine expression.