Starburst amacrine cells (SACs) process complex visual signals in the retina using both acetylcholine (ACh) and gamma-aminobutyric acid (GABA), but the synaptic organization and function of ACh-GABA corelease remain unclear. Here, we show that SACs make cholinergic synapses onto On-Off direction-selective ganglion cells (DSGCs) from all directions but make GABAergic synapses onto DSGCs only from the null direction. ACh and GABA were released differentially in a Ca 2+ level-specific manner, suggesting the two transmitters were released from different vesicle populations. Despite the symmetric cholinergic connection, the light-evoked cholinergic input to a DSGC, detected at both light onset and offset, was motion- and direction-sensitive. This input was facilitated by two-spot apparent motion in the preferred direction but supressed in the null direction, presumably by a GABAergic mechnism. The results revealed a high level of synaptic intricacy in the starburst circuit and suggested differential, yet synergistic, roles of ACh-GABA cotransmission in motion sensitivity and direction selectivity. ► Detected ACh-GABA synaptic cotransmission in the retina ► Discovered symmetric ACh and asymmetric GABA circuits in the starburst network ► Discovered differential regulation of ACh and GABA releases from starburst cells ► Revealed a role of ACh-GAB corelease in motion sensitivity and direction selectivity