A cell line was established from Culex tarsalis Coquillett embryonated eggs and designated as CxTr. The cell line is heterogeneous, composed predominantly of small, round cells, and spindle-shaped cells with a doubling time of approximately 52–60 h. The identity of the cell line was verified as Cx. tarsalis by sequencing of cytochrome oxidase I and the cells were found to be free of contaminating cells, bacteria, fungi, and mycoplasma. The permissiveness of CxTr cells to arbovirus infection was investigated with vaccine and wildtype arboviruses from four viral families: Flaviviridae (Japanese encephalitis virus), Phenuiviridae (Rift Valley fever phlebovirus), Rhabdoviridae (vesicular stomatitis virus), and Togaviridae (Mayaro virus). All viruses were able to infect and replicate within CxTr cells.