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Background Colon cancer is the third most common cancer type worldwide. Novel alternative therapeutic anti-cancer drugs against colon cancer with less toxicity are to be explored . This study was aimed to explore the anti-proliferative and anti-migratory activity of various fractions of Annona reticulata ethanolic leaf extract on human colon cancer cell lines (HCT-116) and to explore the potential molecular targets from the most potent plant extract fraction. Methods After obtaining ethical clearance from the institutional ethics committee, the extract and fractions were prepared and a preliminary analysis of the phytochemical was done qualitatively. Total phenolic and flavonoids were determined. Ethanolic leaf extract and its fractions were subjected to cytotoxicity analysis using the sulforhodamine B assay and the most promising fraction which showed the highest viability was selected to study anti-migratory activity. The anti-migratory effect was studied using a scratch wound healing assay. Gas chromatography-mass spectrometry (GC-MS) was done to identify the major phytocompounds present in the fraction. The major five phytocompounds identified from the GC-MS were subjected to bioinformatics analysis. Result Among the four fractions, the petroleum ether fraction exhibited the highest anti-proliferative activity. The migration of colon cancer cells was significantly inhibited by the extract and petroleum ether fraction. The major phytocompounds identified from GC-MS were phytol (13.03%), 2,6-bis (3,4-methylenedioxyphenyl)-3,7-dioxabicyclo (3.3.0) octane (11.95%), gamma.-sitosterol (10.45%), alpha.-tocopherol-beta.-D-mannoside (7.50%) and 3-amino-4-piperonyl-5-pyrazolone (5.84%). The bioinformatics analysis of these phytochemicals showed a high potential to affect the levels of key proteins driving colon cancer progression, inhibiting the enzymes and proteins overexpressed in cancer. Conclusion The outcome of this study endorses the potential of phytochemicals of the petroleum ether fraction of ethanolic leaf extract of Annona reticulata for the development of a new chemotherapeutic agent in the treatment of colon cancer.