Technical Abstract: Chitinases belong to a large and diverse family of hydrolytic enzymes that break down glycosidic bonds of chitin. However, very little is known about the function of chitinase genes in regulating the chitin content in peritrophic matrix (PM) of the midgut in insects. We identified a cDNA putatively encoding a new chitinase (OnCht) in European corn borer (ECB; Ostrinia nubilalis). The OnCht gene was predominately expressed in larval midgut with no detectable expression in eggs, pupae, or adults. When the larvae were fed an artificial diet, the expression of OnCht increased 4.4-fold but the expression of the gut-specific chitin synthase (OnCHS 2) gene decreased 2.5-fold as compared with those of unfed larvae. In contrast, when larvae were fed and then starved for 24h, the expression of OnCht decreased 1.8-fold but the expression of OnCHS-2 increased 1.8-fold. Furthermore, there was a negative relationship between OnCht gene expression and chitin contents in the midgut. By using a feeding-based RNAi technique, we were able to reduce OnCht transcript level about 63% in the larvae. Consequently, these larvae showed significantly increased chitin content (26%) in the PM but decreased larval body weight (54 %) as compared with control larvae fed on the diet containing GFP dsRNA. We provide strong evidence for the first time that OnCht plays an important role in regulating chitin content of the PM and it effects the growth and development of the ECB larvae.