Compared to the canonical sphingoid backbone of sphingolipids (SLs), atypical long-chain bases (LCBs) lack C1-OH (1-deoxy-LCBs) or C1-CH2OH (1-deoxymethyl-LCBs). In addition, when unsaturated, they present a cis-double bond instead of the canonical Delta 4-5 trans-double bond. These atypical LCBs are directly correlated with the development and progression of hereditary sensory and autonomic neuropathy type 1 and diabetes type II through yet unknown mechanisms. Changes in membrane properties have been linked to the biological actions of SLs. However, little is known about the influence of the LCB structure, particularly 1-deoxy(methyl)-LCB, on lipid-lipid interactions and their effect on membrane properties. To address this question, we used complementary fluorescence-based methodologies to study membrane model systems containing POPC and the different LCBs of interest. Our results show that 1-deoxymethyl-LCBs have the highest ability to reduce the fluidity of the membrane, while the intermolecular interactions of 1-deoxy-LCBs were found to be weaker, leading to the formation of less-ordered domains compared to their canonical counterparts-sphinganine and sphingosine. Furthermore, while the presence of a trans-double bond at the Delta 4-5 position of the LCB increased the fluidity of the membrane compared to a saturated LCB, a cis-double bond completely disrupted the ability of the LCB to segregate into ordered domains. In conclusion, even small changes on the structure of the LCB, as seen in 1-deoxy(methyl)-LCBs, strongly affects lipid-lipid interactions and membrane fluidity. These results provide evidence that altered balance between species with different LCBs affect membrane properties and may contribute to the pathobiological role of these lipids.