MiRP1 (encoded by the KCNE2 gene) is one of a family of five single transmembrane domain voltage-gated potassium (Kv) channel ancillary subunits currently under intense scrutiny to establish their position in channel complexes and elucidate α subunit contact points, but its structure is unknown. MiRP1 mutations are associated with inherited and acquired cardiac arrhythmia. Here, synthetic peptides corresponding to human MiRP1 (full-length and separate domains) were structurally analyzed using FTIR and CD spectroscopy. The N-terminal (extracellular) domain was soluble and predominantly non-ordered in aqueous media, but predominantly α-helical in L-α-lysophosphatidylcholine (LPC) micelles. The MiRP1 transmembrane domain was predominantly a mixture of α-helix and non-ordered structure in LPC micelles, with a minor contribution from non-aggregated β-strand. The intracellular C-terminal domain was insoluble in aqueous solution; reconstitution into non-aqueous environments resulted in solubility and adoption of increasing amounts of α-helix, with the solvent order sodium dodecyl sulphate < dimyristoyl L-α-phosphatidylcholine (DMPC) < LPC < trifluoroethanol. Correlation of secondary structure changes with lipid transition temperature during heating suggested that the MiRP1 C-terminus incorporates into DMPC bilayers. Full-length MiRP1 was soluble in SDS micelles and calculated to contain 34% α-helix, 23% β-strand and 43% non-ordered structure in this environment, as determined by CD spectroscopy. Thus, MiRP1 is highly dependent upon hydrophobic interaction via lipid and/or protein contacts for adoption of ordered structure without nonspecific aggregation, consistent with a role as a membrane-spanning subunit within Kv channel complexes. These data will provide a structural framework for ongoing mutagenesis-based in situ structure-function studies of MiRP1 and its relatives.