Invariant natural killer T (iNKT) cells are a subset of innate-like T cells, which play important regulatory roles in multiple diseases including infection, tumor and metabolic diseases. Revealing the cellular and molecular mechanisms that regulate the development, differentiation and function of iNKT cells is of great significance to elucidate the relationship between iNKT cells and diseases and to seek possible therapeutic approaches. Etoposide-induced protein 2.4 (Ei24) is an autophagy-associated protein which involved in the regulation of cell growth and apoptosis. However, whether Ei24 could regulate iNKT cell differentiation and functions remains unclear. Here, using Cre/loxP system to specifically delete Ei24 in T cells, we found that Ei24 was required for terminal maturation of iNKT cells in thymus, liver and spleen. iNKT1 and iNKT17, but not iNKT2 cells, were affected by Ei24 deficiency. Furthermore, we found that the production of IFN-γ, but not IL-4, was impaired in Ei24 deficient iNKT cells when lipid antigen α-GC was injected in vivo. These results demonstrate that Ei24 is required for the development and function of iNKT cells.