Rana grylio virus (RGV), a member of genus Ranavirus in the family Iridoviridae, is a viral pathogen infecting aquatic animal. RGV 43R has homologues only in Ranavirus and contains a transmembrane (TM) domain, but its role in RGV infection is unknown. In this study, 43R was determined to be associated with virion membrane. The transcripts encoding 43R and the protein itself appeared late in RGV-infected EPC cells and its expression was blocked by viral DNA replication inhibitor, indicating that 43R is a late expressed protein. Subcellular localization showed that 43R-EGFP fusion protein distributed in cytoplasm of EPC cells and that TM domain is essential for its distribution in cytoplasm. 43R-EGFP fusion protein colocalized with viral factories in RGV-infected cells. A recombinant RGV deleting 43R (43R-RGV) was constructed by homologous recombination to investigate its role in virus infection. Compared with wild type RGV, the ability of 43R-RGV to induce the cytopathic effect and its virus titers were significantly reduced. Furthermore, it is revealed that 43R deletion significantly inhibited viral entry but did not influence viral DNA replication by measuring and comparing the DNA levels of RGV and 43R-RGV in the infected cells at the early stage of infection. RGV neutralization with anti-43R serum reduced the virus titer. Therefore, these data showed that RGV 43R is a late gene that encodes an envelope protein involved in RGV entry.