Infection with wild-type Listeria monocytogenes activates a host cytosolic surveillance response characterized by the expression of beta interferon (IFN-β). We performed a genetic screen to identify L. monocytogenes transposon insertion mutants that induced altered levels of host IFN-β expression. One mutant from this screen induced elevated levels of IFN-β and harbored a Tn917 insertion upstream of lmo0558. This study identified lmo0558 as the 6-phosphogluconolactonase gene (pgl), which encodes the second enzyme in the pentose phosphate pathway. pgl mutant L. monocytogenes accumulated and secreted large amounts of gluconate, likely derived from labile 6-phosphogluconolactone, the substrate of Pgl. The pgl deletion mutant had decreased growth in glucose-limiting minimal medium but grew normally when excess glucose was added. Microarray analysis revealed that the pgl deletion mutant had increased expression of several β-glucosidases, consistent with known inhibition of β-glucosidases by 6-phosphogluconolactone. While growth in macrophages was indistinguishable from that of wild-type bacteria, pgl mutant L. monocytogenes exhibited a 15- to 30-fold defect in growth in vivo. In addition, L. monocytogenes harboring an in-frame deletion of pgl was more sensitive to oxidative stress. This study identified L. monocytogenes pgl and provided the first link between the bacterial pentose phosphate pathway and activation of host IFN-β expression.