This study was carried out for the isolation, purification, and characterization of enzyme phospholipase D (PLD) from Amygdalus spinosissima Bunge (wild thorny almond). It was observed that the crude extract showed higher values of enzyme activity 1206 U than that of purified which was found to be 915 U. The optimum time for extraction was 1.5 hours, after which the activity started decreasing. Purification of enzyme was done by hydrophobic interaction chromatography with a specific effect of calcium using Octyl Sepharose. In the absence of calcium ion, the fraction eluted from the column showed high enzymatic activity. The highest specific activity (4575 Umg -1 ), purification fold (53.2) and yield (75.8%) were obtained. Molecular weight was estimated as 62 kDa by SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) analysis. Highest activity of PLD was observed at 50℃ and at 6 pH. Calcium ion stability of PLD activity was found at 40-90 mM. It is concluded from the present study that PLD has numerous industrial applications. It can be extracted and purified from A. spinosissima which is a cheap and easily available source at industrial scale.