The alphaviral 6k gene region encodes the two structural proteins 6K protein and, due to a ribosomal frameshift event, the transframe protein (TF). Here, we characterized the role of the 6k proteins in the arthritogenic alphavirus Ross River virus (RRV) in infected cells and in mice, using a novel 6k in-frame deletion mutant. Comprehensive microscopic analysis revealed that the 6k proteins were predominantly localized at the endoplasmic reticulum of RRV-infected cells. RRV virions that lack the 6k proteins 6K and TF [RRV-(¿6K)] were more vulnerable to changes in pH, and the corresponding virus had increased sensitivity to a higher temperature. While the 6k deletion did not reduce RRV particle production in BHK-21 cells, it affected virion release from the host cell. Subsequent in vivo studies demonstrated that RRV-(¿6K) caused a milder disease than wild-type virus, with viral titers being reduced in infected mice. Immunization of mice with RRV-(¿6K) resulted in a reduced viral load and accelerated viral elimination upon secondary infection with wild-type RRV or another alphavirus, chikungunya virus (CHIKV). Our results show that the 6k proteins may contribute to alphaviral disease manifestations and suggest that manipulation of the 6k gene may be a potential strategy to facilitate viral vaccine development.