Basement membranes (BMs) are resilient polymer structures that surround organs in all animals. Tissues, however, undergo extensive morphological changes during development. It is not known whether the assembly of BM components plays an active morphogenetic role. To study in vivo the biogenesis and assembly of Collagen IV, the main constituent of BMs, we used a GFP-based RNAi method (iGFPi) designed to knock down any GFP-trapped protein in Drosophila. We found with this method that Collagen IV is synthesized by the fat body, secreted to the hemolymph (insect blood), and continuously incorporated into the BMs of the larva. We also show that incorporation of Collagen IV determines organ shape, first by mechanically constricting cells and second through recruitment of Perlecan, which counters constriction by Collagen IV. Our results uncover incorporation of Collagen IV and Perlecan into BMs as a major determinant of organ shape and animal form. Display omitted ► In vivo GFP RNA interference (iGFPi) can be used to knock down GFP-trapped proteins ► Fat body-secreted Collagen IV is incorporated to larval basement membranes (BMs) ► Collagen IV shapes cells and organs by exerting a mechanical constricting tension ► Perlecan, recruited into BMs by Collagen IV, counters Collagen IV constriction