6,6′-Trehalose dioleate (6,6′-TDO) is a glycolipid extensively used for in vivo and in vitro mRNA delivery. This compound effectively encapsulates mRNA encoding hemagglutinin A (HA) from influenza A virus for use within lipid nanoparticles (LNPs), enhancing the secretion of Ifn-γ, Il-2, and Tnf-α from isolated murine spleen cells. When administered via intramuscular injection in mice, LNPs containing 6,6′-TDO predominantly accumulate in the liver and spleen. Unlike LNP formulations without 6,6′-TDO, those containing 6,6′-TDO do not reduce serum levels of blood urea nitrogen (BUN) or elevate levels of lactate dehydrogenase (LDH), troponin I, alanine aminotransferase (ALT), or aspartate aminotransferase (AST). Additionally, these LNPs enhance the serum titers of IgG1 and IgG2a antibodies in mice.