Dengue is the most important mosquito-borne viral disease to humans. Bats are potential reservoirs for flaviviruses, including dengue virus (DENV). In this work, Artibeus jamaicensis bats were inoculated with two serotypes of DENV using different routes. For experimental inoculations (EI) 1 and 2, bats were inoculated subcutaneously or intraperitoneally with DENV-4; for EI-3 bats were inoculated intraperitoneally with DENV-1. Mock inoculated bats were kept as controls. In EI-4, bats were bitten by Aedes aegypti mosquitoes infected with DENV-1 or 4. Reverse transcription-polymerase chain reaction assays in plasma and spleen tissue collected from Day 1 to Days 9-17 after inoculation failed to reveal the presence of viral RNA in any of the samples. No evidence of circulating NS1 or specific anti-DENV IgG was detected in the plasma of the inoculated bats. These results indicate that A. jamaicensis bats are incapable of sustaining dengue virus replication and are unlikely to act as reservoirs for this virus.