UDP-GlcNAc:alpha3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I (encoded by Mgat1) controls the synthesis of hybrid, complex, and paucimannose N-glycans. Mice make hybrid and complex N-glycans but little or no paucimannose N-glycans. In contrast, Drosophila melanogaster and Caenorhabditis elegans make paucimannose N-glycans but little or no hybrid or complex N-glycans. To determine the functional requirement for beta1,2-N-acetylglucosaminyltransferase I in Drosophila, we generated null mutations by imprecise excision of a nearby transposable element. Extracts from Mgat1(1)/Mgat1(1) null mutants showed no beta1,2-N-acetylglucosaminyltransferase I enzyme activity. Moreover, mass spectrometric analysis of these extracts showed dramatic changes in N-glycans compatible with lack of beta1,2-N-acetylglucosaminyltransferase I activity. Interestingly, Mgat1(1)/Mgat1(1) null mutants are viable but exhibit pronounced defects in adult locomotory activity when compared with Mgat1(1)/CyO-GFP heterozygotes or wild type flies. In addition, in null mutants males are sterile and have a severely reduced mean and maximum life span. Microscopic examination of mutant adult fly brains showed the presence of fused beta lobes. The removal of both maternal and zygotic Mgat1 also gave rise to embryos that no longer express the horseradish peroxidase antigen within the central nervous system. Taken together, the data indicate that beta1,2-N-acetylglucosaminyltransferase I-dependent N-glycans are required for locomotory activity, life span, and brain development in Drosophila.