Azotobacter vinelandii contains an iron-regulatory small RNA ArrF whose expression is dependent upon the levels of iron and ferric uptake regulator. The deletion of this ArrF-encoding gene resulted in a 300-fold increase in the production of poly-beta-hydroxybutyrate (PHB), a polymer of industrial importance. This a dagger arrF mutant exhibited wild-type growth and growth-associated PHB production. Limited iron and aeration elevated the PHB production in the mutant as well as wild type. Real-time RT-PCR revealed that phbB, phbA, and phbC were upregulated similar to 61-, 18-, and eightfold, respectively, in the mutant. The phbR transcript of the activator PhbR for this operon was also similar to 11 times more abundant. The analysis of phbR transcript predicted a region of complementarity near its Shine-Dalgarno sequence that could potentially basepair with the conserved region of ArrF. These results suggest that ArrF represses the expression of PhbR in an antisense manner and derepression of this activator in the mutant elevates the expression of phbB, phbA, and phbC, resulting in the PHB overproduction.