Peritrophic membranes (PM) are the first line of defense in the midgut of insects. The structure and function of PMs critically rely on the PM proteins and chitin. To understand the formation and function of PMs, we identified three types of PM proteins from larvae of cabbage looper, Trichoplusia ni, by taking a cDNA cloning approach. The identified PM proteins are chitin binding proteins with unique biochemical and molecular characteristics, including the presence of chitin-binding domains, heavy glycosylation and biased distribution of trypsin/chymotrypsin cleavage sites. Biochemical and physiological analyses of the PM proteins both in vitro and in vivo demonstrated that unique PM proteins play the fundamental roles for the PM formation and function.