Purpose: The purpose of the study was to examine the anticancer properties of a natural chemical entity - juglone molecule - against human HL-60 prom yelocytic leukemia cells along with assessing the effects on normal human monocytes. Juglone molecule was examined for its role in autophagy induction, endogenous ROS production, and inhibition of cell migration and invasion. Methods: Cell viability was evaluated by MTT assay and clonogenic assay was performed to analyse colony formation. Autophagy studies were carried out by transmission electron microscopy (TEM) and western blotting analysis. Mitochondrial morphology was observed through MitoTracker Red CMXRos, and mitochondrial ROS production was assessed through confocal microscopy. The cell invasion and migration was assessed via transwell chambers with and without Matrigel. Results: AMTT assay revealed significant, selective (less cytotoxicity towards normal cells) and dose-dependent inhi- bition of HL-60 leukemia cells and clonogenic assay showed impressive decrease in the number of colonies on increased doses of the molecule. TEM analysis showed formation of autophagosomes and induction of cellular damage. Western blotting assay indicated a significant increase in LC3-I and LC3-II and a slight increase in Beclin-I. Confocal microscopy revealed tremendous increase in ROS concentrations in a dose-dependent manner. Transwell chamber assay revealed significant, dose- dependent inhibition of cell migration and invasion. Conclusion: Juglone induced anticancer effects on promyelocytic HL-60 leukemia cells mediated via autophagy induction, endogenous ROS production, and inhibition of cell migration and invasion, thus indicating that juglone may be a potential lead molecule against HL-60 prom yelocytic leukemia cells.