Leishmania synthesize abundant phosphoglycan-containing molecules made up of [Gal-Man-PO(4)] repeating units, including the surface lipophosphoglycan (LPG), and the surface and secreted proteophosphoglycan (PPG). The vector competence of Phlebotomus duboscqi and Lutzomyia longipalpis sand flies was tested using L. major knockout mutants deficient in either total phosphoglycans (lpg2(-) or lpg5A(-)/5B(-)) or LPG alone (lpg1(-)) along with their respective gene add-back controls. Our results confirm that LPG, the major cell surface molecule of Leishmania promastigotes known to mediate attachment to the vector midgut, is necessary to prevent the loss of infection during excretion of the blood meal remnants from a natural vector, P. duboscqi, but not an unnatural vector, L. longipalpis. Midgut digestive enzymes induced by blood feeding pose another potential barrier to parasite survival. Our results show that 36-72 h after the infective feed, all parasites developed well except the lpg2(-) and lpg5A(-)/5B(-) mutants, which showed significantly reduced survival and growth. Protease inhibitors promoted the early survival and growth of lpg2(-) in the blood meal. PPG was shown to be the key molecule conferring resistance to midgut digestive enzymes, as it prevented killing of lpg2(-) promastigotes exposed to midgut lysates prepared from blood-fed flies. The protection was not associated with inhibition of enzyme activities, but with cell surface acquisition of the PPG, which appears to function similar to mammalian mucins to protect the surface of developing promastigotes against proteolytic damage.