Fibrillins play a critical role in tissue/organ development and cardiopulmonary function. Here we showed that O-glucosylation of the fibrillins’ epidermal growth factor-like repeats by protein O-glucosyltransferase 2 (POGLUT2) and POGLUT3 was indispensable for in vivo function of mouse fibrillins. Loss of O-glucosylation of fibrillins caused neonatal death with cardiopulmonary, skeletal, and eye defects reminiscent of fibrillin/elastin mutations. Analyses of FBNs in Poglut2/3 double knockout (DKO) lung, and from DKO dermal fibroblast medium and matrix, provided overwhelming evidence that fibrillins were more susceptible than other POGLUT2/3 substrates to loss of O-glucose. In the DKO, defects in microfibril structure impaired elastic fiber formation, reduced TGF-β/pERK signaling, caused structural defects in late gestation lung blood vessels and terminal bronchioles, and impaired cell differentiation and primary septation in the saccules/alveoli. Collectively, these data support an essential role for POGLUT2/3-mediated O-glucosylation in fibrillin trafficking, microfibril assembly/stability, and function in the ECM environment during lung development.