Abstract Selenoprotein I (SELENOI) is a type I CDP-alcohol phosphatidyltransferase that synthesizes phosphatidylethanolamine (PE) in the final reaction of the Kennedy pathway. We have found that SELENOI deletion in the whole animal is embryonic lethal, which emphasizes the key role PE synthesis plays in cellular division and development. One important role for PE in cells is the role it plays as a ligand for the conversion of LC3I to the lipidated LC3II molecule, which in turn allows the progression of early autophagosomes to proceed to later stage autophagosomes. Using a T cell-specific knockout (KO) model of SELENOI, we investigated SELENOI loss-of-function studies in these immune cells. In particular, SELENOI KO T cells were stimulated with STING-dependent and STING-independent agonists to test the importance of this selenoenzyme in autophagy and downstream signaling events that depend on efficient autophagy. Our results found that PE levels were lower in TCR-activated T cells lacking SELENOI compared to wild-type (WT) controls. LC3I to LC3II conversion was impaired in SELENOI KO T compared to controls, and STING-dependent autophagosome formation was particularly decreased in the absence of SELENOI. Using bone marrow-derived macrophages (BMDMs) from a doxycycline-inducible transgenic mouse that expresses a short hairpin RNA (shRNA) that lowers SELENOI <50%, we found impaired LC3I to LC3II conversion as well as reduced autophagosome formation but not to the extent of that found with the KO T cell model. Our data suggest that SELENOI is an important enzyme for proper lipidation of LC3-I and autophagosome formation within immune cells.