Human phospholipid scramblase 1 (hPLSCR1) belongs to the ATP-independent class of phospholipid translocators which possess a single EF-hand-like Ca 2+-binding motif and also a C-terminal helix (CTH). The CTH domain of hPLSCR1 was believed to be a putative single transmembrane helix at the C-terminus. Recent homology modeling studies by Bateman et al. predicted that the hydrophobic nature of this helix is due to its packing in the core of the protein domain and proposed that this is not a true transmembrane helix [Bateman A, Finn RD, Sims PJ, Wiedmer T, Biegert A & Johannes S. Bioinformatics 2008, 25, 159]. To determine the exact function of the CTH of hPLSCR1, we deleted the CTH domain and determined: (a) whether CTH plays any role beyond membrane anchorage, (b) the functional consequences of CTH deletion, and (c) any conformational changes associated with CTH in a lipid environment. In vitro reconstitution studies confirm that the predicted CTH is required for membrane insertion and scrambling activity. CTH deletion caused a 50% decrease in binding affinity of Ca2+ for CTH-hPLSCR1 (Ka = 115 μm) compared with hPLSCR1 (Ka = 249 μm). Far UV-CD studies revealed that the CTH peptide adopts α-helicity only in the presence of SDS micelles and negatively charged vesicles, indicating that electrostatic interactions are required for insertion of the peptide. CTH peptide-quenching studies confirm that the predicted CTH inserts into the membrane and its ability to interact with the membrane depends on the presence of charge interactions. TOXCAT assay revealed that CTH of hPLSCR1 does not oligomerize in the membrane. We conclude that CTH is required for membrane insertion and Ca2+ coordination and also plays an important role in the functional conformation of hPLSCR1. Phospholipid scramblases constitute a family of homologous proteins named as hPLSCR1-hPLSCR4. The C-terminal helix (CTH) is presently close to the EF-hand motif in the scramblase homologues indicating that it may play an important role in Ca2+ binding. This study shows that CTH is required for membrane insertion, Ca2+ co-ordination and also plays an important role in the functional conformation of hPLSCR1. © 2013 FEBS.