The membrane-associated RING-CH (MARCH) family of proteins are membrane-embedded E3 ubiquitin ligases that regulate the cell surface expression of substrates via ubiquitin-mediated endocytosis and lysosomal degradation. While a diverse pool of cell surface proteins have been identified as MARCH substrates, such as MHC- class I and class II molecules, T cell co-receptor CD4, the NK cell ligand Mult1, and transferrin receptor, the mechanisms by which MARCH recognises these substrates have not been fully elucidated. A common feature among MARCH proteins are transmembrane domains that, in addition to anchoring them to the membrane, have been shown to contribute to substrate preferences. However, no ‘recognition motifs’ within the TM domains of MARCH or substrate have been identified, and the determinants of substrate specificity have yet to be clearly defined. In this study, we employed parallel biochemical and biophysical approaches to characterise the molecular and structural features of the two TM domains of the mammalian MARCH9, and identify important motifs that are required for target down-regulation. Cell-based in vitro assays were used to show that substrate preferences of MARCH9 track with the TM domains of specific substrate molecules. Mutagenesis analysis of TM domains of MARCH9 revealed important sequence elements within this region that are essential for maintaining MARCH9 function against two unrelated substrates. Solution nuclear magnetic resonance (NMR) spectroscopy was used to define the limits of α-helical secondary structure of the TM domains of MARCH9, and confirmed that the sequence elements identified were indeed located in the membrane-embedded regions of MARCH9. These results represent the most comprehensive analysis of the TM domains of MARCH9 to date, and provide a rationale for more detailed studies of the TM domains of the other MARCH proteins. Understanding the mechanisms by which MARCH9 TM-domain mutants malfunction will provide new insights into how MARCH proteins recognise target molecules.