Phospholipid scramblases (PLSCRs) constitute a group of homologous bidirectional lipid translocators that are conserved in all eukaryotic organisms. In humans, four related PLSCR genes have been identified, named hPLSCR1- hPLSCR4. The first described member and prototype of this family is hPLSCR1, a 37 kDa type II endofacial membrane protein, that is multipalmitoylated and widely expressed in most human tissues. hPLSCR1 is involved in the rapid calcium dependent translocation of plasma membrane phospholipids, although neither the detailed calcium-induced conformational change nor the mechanism of phospholipid scrambling are known yet. In addition to this role hPLSCR1 may also function in regulating processes including signaling, cell differentiation, apoptosis, injury, cell proliferation and transcription.In the present contribution we have studied the role of the C-terminal α-helix (30 aa residues) in the structure-function relationship of hPLSCR1. With that aim a truncated mutant was constructed lacking the C-terminal α-helix (hPLSCR1αC290). A combination of structural and functional studies (fluorescence and infrared spectroscopies, partial trypsin digestion and functional characterization using liposomes) reveal that the α-helix is crucial for the scramblase activity. Furthermore in the presence of calcium the truncated mutant displays a much lower affinity for this ion and, although it still undergoes conformational changes, it requires higher Ca2+ concentrations than the wild type. Calcium binding increases the truncated mutant stability, inducing protection against trypsin digestion and thermal denaturation.