Natural antisense transcripts (NATs) are naturally widespread and have very important functions in regulating expression of their target genes. In this report, we identified a novel NAT, designated mucD-AS, transcribed from the opposite strand ofmucD gene locus, which has a pivotal function in regulating the biosynthesis of alginate. For the biotechnological and medical significance in the study of alginate biosynthesis, we investigated the effect ofmucD-AS on mucD expression and alginate biosynthesis, and confirmed mucD-AS can induce biofilm formation ofP. aeruginosa significantly. Since alginate has a key function in the infection process and acts as virulence factor of P. aeruginosa, we speculate mucD-AS may have a regulatory function in this process.