Circadian behavioral patterns in mosquitoes can be observed through their locomotor activity, which includes fundamental behaviors such as foraging, mating, and oviposition. These habits, which are fundamental to the life cycle of Anopheles mosquitoes, are closely related to pathogen transmission to humans. While rhythmic cycles of locomotor activity have been described in Anopheles species, no studies have been conducted on Anopheles darlingi species, the main malaria vector in the Amazon region. The aim of this study was to investigate how insemination status, blood meal, and Plasmodium vivax infection affect the locomotor activity of An. darlingi. The experiments were performed with 3- to 10-day-old An. darlingi females, which had been fed with 15% honey solution. These mosquitoes were obtained from the Malaria Vector Production and Infection Platform (PIVEM)/FIOCRUZ–RO. The experimental groups were divided into four categories: virgin vs. inseminated, unfed virgin vs. blood-fed virgin, unfed inseminated vs. blood-fed inseminated, and infected blood vs. uninfected blood. Locomotor activity was monitored using the Flybox equipment, capturing images that were subsequently converted into video to measure the insect activity, using PySoLo software. The periodicity and rhythmicity of mosquito locomotor activity were analyzed using MatLab® software. The locomotor activity of An. darlingi females showed a nocturnal and bimodal pattern under LD conditions. When comparing the insemination states and blood meal, there was a reduction in the locomotor activity in inseminated and blood-fed females. However, the P. vivax⁺ infection did not increase locomotor activity of An. darlingi species.