Studies on Culex tarsalis Coquillett in Colorado have shown marked seasonal variation in the proportion of blood meals from birds and mammals. However, limitations in the specificity of antibodies used in the precipitin test and lack of vertebrate host availability data warrant revisiting Cx. tarsalis blood feeding behavior in the context of West Nile virus (WNV) transmission. We characterized the host preference of Cx. tarsalis during peak WNV transmission season in eastern Colorado and estimated the relative contribution of different avian species to WNV transmission. Cx. tarsalis preferred birds to mammals each month, although the proportion of blood meals from mammals increased in July and August. The distribution of blood meals differed significantly across months, in part because of changes in the proportion of blood meals from American robins, a preferred host. The estimated proportion of WNV-infectious vectors derived from American robins declined from 60 to 1% between June and August. The majority of avian blood meals came from doves, preferred hosts that contributed 25–40% of the WNV-infectious mosquitoes each month. Active WNV transmission was observed in association with a large house sparrow communal roost. These data show how seasonal patterns in Cx. tarsalis blood feeding behavior relate to WNV transmission in eastern Colorado, with the American robin contributing greatly to early-season virus transmission and a communal roost of sparrows serving as a focus for late-season amplification.