West Nile virus (WNV), a mosquito-transmitted single-stranded RNA (ssRNA) flavivirus, causes human disease of variable severity. We investigated Toll-like receptor 7-deficient (Tlr7(-/-)) and myeloid differentiation factor 88-deficient (Myd88(-/-)) mice, which both have defective recognition of ssRNA, and found increased viremia and susceptibility to lethal WNV infection. Despite increased tissue concentrations of most innate cytokines, CD45(+) leukocytes and CD11b(+) macrophages failed to home to WNV-infected cells and infiltrate into target organs of Tlr7(-/-) mice. Tlr7(-/-) mice and macrophages had reduced interleukin-12 (IL-12) and IL-23 responses after WNV infection, and mice deficient in IL-12 p40 and IL-23 p40 (Il12b(-/-)) or IL-23 p19 (Il23a(-/-)), but not IL-12 p35 (Il12a(-/-)), responded similarly to Tlr7(-/-) mice, with increased susceptibility to lethal WNV encephalitis. Collectively, these results demonstrate that TLR7 and IL-23-dependent WNV responses represent a vital host defense mechanism that operates by affecting immune cell homing to infected target cells.