The recombinant dengue virus type 4 (rDEN4) vaccine candidate, rDEN4Δ30, was found to be highly infectious, immunogenic and safe in human volunteers. At the highest dose (105 PFU) evaluated in volunteers, 25% of the vaccinees had mild elevations in liver enzymes that were rarely seen at lower doses. Here, we describe the generation and selection of additional mutations that were introduced into rDEN4Δ30 to further attenuate the virus in animal models and ultimately human vaccinees. Based on the elevated liver enzymes associated with the 105 PFU dose of rDEN4Δ30 and the known involvement of liver infection in dengue virus pathogenesis, a large panel of mutant viruses was screened for level of replication in the HuH-7 human hepatoma cell line, a surrogate for human liver cells and selected viruses were further analyzed for level of viremia in SCID-HuH-7 mice. It was hypothesized that rDEN4Δ30 derivatives with restricted replication in vitro and in vivo in HuH-7 human liver cells would be restricted in replication in the liver of vaccinees. Two mutations identified by this screen, NS3 4995 and NS5 200,201, were separately introduced into rDEN4Δ30 and found to further attenuate the vaccine candidate for SCID-HuH-7 mice and rhesus monkeys while retaining sufficient immunogenicity in rhesus monkeys to confer protection. In humans, the rDEN4Δ30-200,201 vaccine candidate administered at 105 PFU exhibited greatly reduced viremia, high infectivity and lacked liver toxicity while inducing serum neutralizing antibody at a level comparable to that observed in volunteers immunized with rDEN4Δ30. Clinical studies of rDEN4Δ30-4995 are ongoing.