The insect midgut is generally lined with a unique protective chitin/protein structure, the peritrophic membrane (PM). We demonstrated that in Trichoplusia ni larvae, the majority of PM proteins were assembled with chitin as a consequence of their chitin binding properties. These proteins could be dissociated from the PM in vitro by Calcofluor, a well-known chemical with chitin binding properties. The chitin binding characteristics of PM proteins were confirmed by their high affinity binding in vitro to regenerated chitin. In vivo assays demonstrated that Calcofluor could inhibit PM formation in five lepidopteran insects tested. The inhibition of T. ni PM formation by Calcofluor, was accompanied by increased larval susceptibility to baculovirus infection. Continuous inhibition of PM formation by Calcofluor resulted in retarded larval development and mortality. The destructive effect of Calcofluor on PM formation was demonstrated to be transient and reversible depending on the presence of Calcofluor within the midgut. In addition, degradation of the insect intestinal mucin was observed concurrently with the inhibition of PM formation by Calcofluor. Our studies revealed a potential novel approach to develop strategies for insect control by utilizing chitin binding molecules to specifically target PM formation in a broad range of insect pest species.