Visceral leishmaniasis (VL) caused by Leishmania donovani is widespread in Ethiopia, particularly in the north and north-west of the country. Studies to investigate the ecology and behavior of sandflies and their epidemiological significance in the transmission of VL were conducted in Tahtay Adiyabo district, northern Ethiopia. Entomological studies were undertaken in three villages of the district between May 2011 and April 2012 to identify the sandfly fauna and determine the bionomics of Phlebotomus orientalis. Collections of sandflies were done using CDC light traps, sticky traps, and pyrethrum spray catches inside residential huts. The vectorial role of sandfly vector (s) was determined by the detection of Leishmania parasites by microscopy and Leishmania specific PCR. The host preferences of P. orientalis was examined using host choice experiments and bloodmeal analysis using cytochrome (cyt) b-PCR and reverse line blotting as well as enzyme linked immunosorbent assay. Nocturnal periodicity of P. orientalis was also investigated using light traps by replacing the collection bags at hourly intervals throughout the night. The attractiveness of nine plant species for P. orientalis was also done under field settings using unlit CDC traps. The effects of lunar phases and lunar periodicity on the performance of light traps in collecting P. orientalis were studied by sampling sandflies among the four lunar phases for seven months. In total, 100,772 sandflies, belonging to 25 species were recorded. Sergentomyia africana and P. orientalis made up 59.1% and 23.5% of the collected sandflies, respectively. The outdoor to indoor index for P. orientalis was 138:1 on sticky traps, exhibiting its pronounced exophilic behavior. P. orientalis showed marked fluctuations in seasonal density, which peaked during the months of March and April. A sharp decrease in abundance of P. orientalis was also observed from July to December. The parous rate in the unfed females was 34.05% in peri-domestic and 35.35% in agricultural fields. Out of 921 females of P. orientalis dissected, one specimen (0.11%) was found naturally infected with Leishmania promastigotes. Five pools (25 females) of unfed P. orientalis had DNA of Leishmania spp. Markedly higher mean numbers of female P. orientalis were attracted to donkey-baited tent traps than traps-baited with cow, human, dog, goat, sheep or chicken, respectively. Among the small wild animalsrnivrntested, ground squirrels attracted significantly (P