Consideringthegreatpotentialofnaturalproductsasanticanceragents,thepresentstudywasdesignedtoexplorethemolecularmechanismsresponsibleforanticanceractivitiesofMesuaferreastembarkextractagainsthumancolorectalcarcinoma.BasedonMTTassayresults,bioactivesub-fraction(SF-3)wasselectedforfurtherstudiesusingHCT116cells.Repeatedcolumnchromatographyresultedinisolationoflessactiveα-amyrinfromSF-3,whichwasidentifiedandcharacterizedbyGC-MSandHPLCmethods.α-amyrinandbetulinicacidcontentsofSF-3weremeasuredbyHPLCmethods.Fluorescentassaysrevealedcharacteristicapoptoticfeatures,includingcellshrinkage,nuclearcondensation,andmarkeddecreaseinmitochondrialmembranepotentialinSF-3treatedcells.Inaddition,increasedlevelsofcaspases-9and-3/7levelswerealsoobservedinSF-3treatedcells.SF-3showedpromisingantimetastaticpropertiesinmultipleinvitroassays.Multi-pathwayanalysisrevealedsignificantdown-regulationofWNT,HIF-1α,andEGFRwithsimultaneousup-regulationofp53,Myc/Max,andTGF-βsignallingpathwaysinSF-3treatedcells.Inaddition,promisinggrowthinhibitoryeffectswereobservedinSF-3treatedHCT116tumourspheroids,whichgiveahintaboutinvivoantitumorefficacyofSF-3phytoconstituents.Inconclusion,theseresultsdemonstratedthatanticancereffectsofSF-3towardscoloncancerarethroughmodulationofmultiplemolecularpathways.