GTPases are molecular switches, which regulate a variety of cellular processes such as cell polarity, gene transcription, microtubule dynamics, cell-cycle etc. In this paper, we characterize a Ca2+-binding protein from Entamoeba histolytica (EhCaBP6) as a novel GTPase. We locate the active site for GTP hydrolysis within the C-terminal domain of EhCaBP6, although it requires full length protein for its complete range of activity. Using NMR studies, we observe that GTP binding induces conformational change in EhCaBP6. The identification of this novel and unusual Ca2+-dependent GTPase is important to elucidate the unconventional cell cycle of E. histolytica. • We characterize a Ca2+-binding protein (EhCaBP6) from Entamoeba histolytica as a novel GTPase. • C-terminal domain of EhCaBP6 possesses the active site, as identified by mutational studies, for GTP binding and hydrolysis. • GTPase activity together with its involvement in the cell cycle, establishes EhCaBP6 as a novel cell cycle regulating GTPase. [ABSTRACT FROM AUTHOR]