The present study was taken up to evaluate the apoptosis inducing ability of alcoholic extract of whole plant of Anagallis arvensis (AAE) in HL-60 cells. We observed time and concentration dependent decrease in cell viability after treatment with AAE. Fluorescent staining and scanning electron micrographs of treated HL-60 cells demonstrated chromatin condensation, nuclear fragmentation and formation of apoptotic blebs. There was a marked increase in hypodiploid population of cells as observed by cell cycle analysis. Annexin V-FITC/PI also depicted the presence of apoptotic cells. Anti-apoptotic protein Bcl-2 was observed to be decreased by 62% at 20 µg/ml concentration and a significant increase in ROS production up to 6.9-fold was observed in time dependent manner. In addition, alteration in mitochondrial membrane potential was observed, which was followed by cytochrome c release to cytoplasm. Activated levels of mitochondrial downstream pathway protein namely Caspase-3 and 9, were detected in treated HL-60 cells by colorimetric analysis. DNA ladder formation, a biochemical hallmark of apoptosis was also observed in treated HL-60 cells. The results of the present study support the apoptotic potential of AAE and probability of its promising role in development as effective anticancer agent against leukemia cells.