Objective Acetylcholine is a highly conserved neurotransmitter that plays a crucial role in the regulation of animal motor behavior. Abnormalities in acetylcholine signaling can lead to various motor dysfunctions. However, the inhibitory regulatory mechanisms of acetylcholine in motor behavior are not fully understood. In this study, we used Caenorhabditis elegans as a model organism to investigate the regulatory effects of acetylcholine-gated chloride channel receptor subunits (ACC-1, ACC-2, ACC-3, ACC-4) on motor behavior.Methods We used a combination of locomotion tracking, molecular genetics, and optogenetics to analyze C. elegans locomotion in acetylcholine-gated chloride channel subunit deficient mutants.Results We found that mutations in these subunits affected the kinematics of forward, backward, and turning movements of nematodes. The body bending amplitude during forward movement was also modified. Optogenetic activation of RIB interneurons led to delayed termination of the reversal in these mutant strains.Conclusion These results suggest that the regulation of acetylcholine-gated chloride channel subunits is required for maintaining and modulating C. elegans motor states. They also suggest that these subunits may be involved in mediating the inhibitory regulation of RIB interneurons on backward movement in C. elegans. This study provides new insights into the regulatory mechanisms of acetylcholine-gated inhibitory receptors in motor behavior.