The present study investigated mosquito species composition and phenotypic insecticide resistance profile to support decision-making in vector control in the Republic of Djibouti at the Horn of Africa. Adult mosquitoes were collected between December 2016 and December 2017 across 20 sentinel sites established in the 6 regions of the country using both Centers for Disease Control (CDC) miniature light traps and pyrethrum spray catches (PSC). Female mosquitoes were kept aside, for morphological identification to species by an expert entomologist using appropriate taxonomic keys by Gillies & Coetzee and Glick. Bioassays were also conducted in An. stephensi from Djibouti-ville against nine insecticides used in public health. A total number of 12,538 host-seeking mosquitoes belonging to four genera (Anopheles, Culex, Aedes, Uranotaenia) comprising 12 species were collected. Among these, A. gambiae S.L. and A. stephensi were the two major malaria vectors identified while secondary malaria vectors such as A. nili somalicus, A. dthali and A. azaniae were also collected. Culex quinquefasciatus was the most abundant mosquito species in the 6 regions. WHO susceptibility tests performed on A. stephensi population from Djibouti-ville showed resistance to pyrethroids, organophosphates, carbamates and DDT. The resistance intensity bioassays indicated low to moderate intensity of resistance with pyrethroid insecticides and the organophosphate pirimiphos methyl. Meanwhile pre-exposure to PBO suggested involvement of P450 detoxification enzymes in pyrethroid resistance. These findings revealed the urgent need to develop and implement a programme for monitoring and managing insecticide resistance in local vector populations with efficient control strategies in Djibouti.