Acetylcholinergic dysfunction is a key underlying feature in the cognitive decline associated with aging. The machinery required for presynaptic acetylcholine (ACh) release and post-synaptic signaling have been described in detail, yet the precise relationship between alterations in ACh neurotransmission and downstream changes in behavior in the nervous system remain poorly understood. The vesicular acetylcholine transporter (VAChT) is a critical element of cholinergic signaling, mediating the transport of ACh from the cytoplasm into synaptic vesicles for exocytotic release. A complete loss of Vacht is lethal in flies, worms and mammals. Here we hypothesize that subtle changes in Vacht will uncover important roles of ACh release in behavior. Using point mutations, recovered in an ethyl methanesulfonate screen, to disrupt VAChT function, we report that a series of Vacht mutations impair two locomotion circuits, baseline and touch response locomotion. In particular, Vacht mutants display a deficit in the timing of the response to touch stimulus in a manner corresponding to severity of the mutant alleles studied. We categorized the resulting allelic series as mild, moderate and severe with respect to acetylcholine release. We further report the genetic rescue of Vacht mutant deficits using wildtype VAChT. In addition, we conducted a pharmacological rescue study using agonists of the dopaminergic and cholinergic pathways, and found that both drugs partly rescued the locomotion deficits in the mutants. Together, this report supports a key role for acetylcholine signaling in regulating the timing of the response to a mechanical stimulus and underscores the utility of point mutations that compromise VAChT activity in vivo as tools to elucidate the complex relationship between altered ACh release and behavioral deficits.