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Background: Malaria is a great public health burden and Africa suffers the largest share of malaria-attributed deaths. Despite control efforts targeting indoor malaria transmission, such as insecticide-treated bed nets (ITNs) and deployment of indoor residual spraying, transmission of the parasite in western Kenya is still maintained. This study was carried out to determine the impact of ITNs on indoor vector densities and biting behaviour in western Kenya. Methods: Indoor collection of adult mosquitoes was done monthly in six study sites in western Kenya using pyrethrum spray collections from 2012 to 2014. The rotator trap collections were done in July-August in 2013 and May-June in 2014. Mosquitoes were collected every 2 h between 18.00 and 08.00 h. Human behaviour study was conducted via questionnaire surveys. Species within Anopheles gambiae complex was differentiated by PCR and sporozoite infectivity was determined by ELISA. Species distribution was determined and bed net coverage in the study sites was recorded. Results: During the study a total of 5,469 mosquito vectors were collected from both PSC and Rotator traps comprising 3,181 (58.2%) Anopheles gambiae and 2,288 (41.8%) Anopheles funestus. Compared to all the study sites, Rae had the highest density of An. gambiae with a mean of 1.2 (P < 0.001) while Kombewa had the highest density of An. funestus with a mean of 1.08 (P < 0.001). Marani had the lowest density of vectors with 0.06 An. gambiae and 0.17 An. funestus (P < 0.001). Among the 700 PCR confirmed An. gambiae s.l. individuals, An. gambiae s.s. accounted for 49% and An. arabiensis 51%. Over 50% of the study population stayed outdoors between 18.00 and 20.00 and 06.00 and 08.00 which was the time when highest densities of blood fed vectors were collected. Anopheles gambie s.s. was the main malaria parasite vector in the highland sites and An. arabiensis in the lowland sites. Bed net ownership in 2012 averaged 87% across the study sites. Conclusions: This study suggests that mass distribution of ITNs has had a significant impact on vector densities, species distribution and sporozoite rate. However, shift of biting time poses significant threats to the current malaria vector control strategies which heavily rely on indoor controls. [ABSTRACT FROM AUTHOR]