Inhibitory GABAergic transmission is required for proper circuit function in the nervous system. However, our understanding of molecular mechanisms that preferentially influence GABAergic transmission, particularly presynaptic mechanisms, remains limited. We previously reported that the ubiquitin ligase EEL-1 preferentially regulates GABAergic presynaptic transmission. To further explore how EEL-1 functions, here we performed affinity purification proteomics using Caenorhabditis elegans and identified the O-GlcNAc transferase OGT-1 as an EEL-1 binding protein. This observation was intriguing, as we know little about how OGT-1 affects neuron function. Using C. elegans biochemistry, we confirmed that the OGT-1/EEL-1 complex forms in neurons in vivo and showed that the human orthologs, OGT and HUWE1, also bind in cell culture. We observed that, like EEL-1, OGT-1 is expressed in GABAergic motor neurons, localizes to GABAergic presynaptic terminals, and functions cell-autonomously to regulate GABA neuron function. Results with catalytically inactive point mutants indicated that OGT-1 glycosyltransferase activity is dispensable for GABA neuron function. Consistent with OGT-1 and EEL-1 forming a complex, genetic results using automated, behavioral pharmacology assays showed that ogt-1 and eel-1 act in parallel to regulate GABA neuron function. These findings demonstrate that OGT-1 and EEL-1 form a conserved signaling complex and function together to affect GABA neuron function. GABA neurons are a critical component of nervous systems across the animal kingdom from mammals (1, 2) to simple invertebrates, such as Caenorhabditis elegans (3, 4). They provide essential inhibitory activity within neural circuits. In humans, various dysfunctions in GABA neurons and the imbalance between excitatory and inhibitory neurotransmission contribute to neurodevelopmental disorders (5, 6). Thus, understanding how GABA neuron function is regulated is critical for our understanding of nervous system function and disease. Much remains unknown about molecular mechanisms that preferentially affect GABAergic transmission. Core presynaptic machinery, such as synaptotagmin, the SNARE complex, and active zone proteins, influence both glutamatergic and GABAergic transmission (7, 8). A few post-synaptic regulators that preferentially or specifically affect GABAergic transmission are known, including Gephyrin, Neuroligin2, Slitrk3, and GARHLs (9-13). In mammals, less is known about presynaptic GABA-specific regulators, but some proteins, such as synapsins, can differentially impact inhibitory transmission compared with excitatory transmission (14, 15). In C. elegans, core presynaptic components play conserved roles in neurotransmission in the motor circuit, a model circuit with balanced excitatory cholinergic and inhibitory GABAergic neuron function (4, 16). Like mammals, relatively few proteins are known that preferentially regulate presynaptic GABA function in C. elegans. Nonetheless, the worm motor circuit has proven valuable for identifying mol...