Background: Malaria remains a major public health concern. Vector control measures based solely on insecticide treated nets (ITNs) and indoor residual spraying (IRS) have demonstrated not to be feasible for malaria elimination. It has been shown that ivermectin affects several aspects of Anopheles species biology. Along the Latin American seacoast, Anopheles aquasalis Curry plays an important role in malaria transmission. The observation of mosquitoes locomotor activity under laboratory conditions can reveal details of their daily activity rhythms, which is controlled by an endogenous circadian clock that seems to be influenced by external signals, such as light and temperature. In this study, we assessed basal locomotor activity and the effects of ivermectin on locomotor activity of the American malaria vector, An. aquasalis. Methods: Adult females of Anopheles aquasalis used in experiments were three to five days post-emergence. Blood from one single subject was used to provide mosquito meals by membrane feeding assays. Powdered ivermectin compound was used to achieve different concentrations of drug as previously described. Fully engorged mosquitoes were individually placed into glass tubes and provided with 10% sucrose. Each tube was placed into a Locomotor Activity Monitor (LAM). The LAMs were kept inside an incubator under a constant temperature and a 12:12 h light:dark cycle. The average locomotor activity was calculated as the mean number of movements performed per mosquito in the period considered. Intervals of time assessed were adapted from a previous study. One-way ANOVA tests were performed in order to compare means between groups. Additionally, Dunnett’s method was used for post-hoc pairwise means comparisons between each group and control. Stata software version 13 was used for the analysis. Results: Anopheles aquasalis showed a nocturnal and bimodal pattern for mosquitoes fed both control blood meals and sub-lethal concentrations of ivermectin. In this species, activity peaks occurred at the beginning of the photophase and scotophase in the control group. The nocturnal activity is evident and higher just after the evening peak and maintains basal levels of locomotion throughout the scotophase. In the entire group analysis, locomotor activity means of experimental sets were significantly lower than control for each period of time evaluated. In the survival group, the locomotor activity means of all treatment sets were lower than control mosquitoes for all intervals of time when both the whole period and scotophase were assessed. When the middle of scotophase was evaluated, means were significantly lower for LC15 and LC25, but not LC5. For the beginning of photophase period, significant differences were detected only between control and LC5. When both the photophase and scotophase were assessed alone, no significant differences were found. Mean locomotor activity was significantly lower for dead group when compared to survival group for all experimental sets when whole period, photophase, and scotophase were assessed. Conclusions: Ivermectin seems to decrease locomotor activity of An. aquasalis at sub-lethal concentrations. The effects on locomotor activity increase according at higher ivermectin concentrations and are most evident during the whole scotophase as well as in the beginning and in the end of this phase, and sub-lethal effects may still be observed in the photophase. Findings presented in this study demonstrate that sub-lethal ivermectin effects reduce mosquito locomotor activity, which could diminish vectorial capacity and therefore the malaria transmission.