The virus is mostly maintained in birds (in the Western hemisphere, particularly the American Robin). Female mosquitoes, mainly of the species Culex pipiens, Culex restuan, and Culex quinquefasciatus, bite infected birds, carry the virus in their salivary glands, and infect other birds when they bite again. Culex pipiens is thought to be the main mosquito species which transmits the virus from birds to mammals. In mammals the virus does not multiply as readily, and it is believed that mosquitoes biting infected mammals do not further transmit the virus. A 2004 paper in Science found that Culex pipiens mosquitoes existed in two populations in Europe, one which bites birds and one which bites humans. In North America 40% of Culex pipiens were found to be hybrids of the two types which bite both birds and humans, providing a vector for West Nile virus. This is thought to provide an explanation of why the West Nile disease has spread more quickly in North America than Europe.

It was initially believed that direct human-to-human transmission was impossible, but in 2002 the Centers for Disease Control and Prevention (CDC) discovered the transmission of West Nile virus through blood transfusion and organ transplants as well as through breast milk, prenatal infection, and occupational exposure. Blood banks in the US now routinely screen for the virus amongst their donors during the epidemic season. In Britain, as a precautionary measure, the National Blood Service runs a test for this disease in donors who donate within 28 days of a visit to the United States or Canada.

There is no vaccine for humans. A vaccine for horses based on killed viruses exists; some zoos have given this vaccine to their birds, although its effectiveness there is unknown.

A 2006 study in Journal of Experimental Medicine found a genetic factor which appears to increase susceptibility to West Nile disease. A mutation of the gene CCR5 gives protection against the HIV virus but leads to more serious complications of WNV infection. Carriers of two mutated copies of CCR5 made up 4 to 4.5% of a sample of West Nile disease sufferers but the incidence of the gene in the general population is only 1%.