Plant apyrases are nucleoside triphosphate (NTP) diphosphohydrolases (NTPDases) and have been implicated in an array of functions within the plant including the regulation of extracellular ATP. Arabidopsis encodes a family of seven membrane bound apyrases (AtAPY1–7) that comprise three distinct clades, all of which contain the five conserved apyrase domains. With the exception of AtAPY1 and AtAPY2, the biochemical and the sub-cellular characterization of the other members are currently unavailable. In this research, we have shown all seven Arabidopsis apyrases localize to internal membranes comprising the cis-Golgi, endoplasmic reticulum (ER) and endosome, indicating an endo-apyrase classification for the entire family. In addition, all members, with the exception of AtAPY7, can function as endo-apyrases by complementing a yeast double mutant (Δynd1Δgda1) which lacks apyrase activity. Interestingly, complementation of the mutant yeast using well characterized human apyrases could only be accomplished by using a functional ER endo-apyrase (NTPDase6), but not the ecto-apyrase (NTPDase1). Furthermore, the substrate specificity analysis for the Arabidopsis apyrases AtAPY1–6 indicated that each member has a distinct set of preferred substrates covering various NDPs (nucleoside diphosphates) and NTPs. Combining the biochemical analysis and sub-cellular localization of the Arabidopsis apyrases family, the data suggest their possible roles in regulating endomembrane NDP/NMP (nucleoside monophosphate) homoeostasis.