To study the mode of action of salinomycin for its anticancer activity, by analysing salinomycin inter actions with DNA, potassium channel and Wnt signalling proteins. PatchDock was used to study the Protein-Ligand in teractions initially and detailed analysis were performed with AutoDock 4.2.1. DNA-Ligand interaction were also p erformed with AutoDock. The docking results were interpreted using PyMol and LigPlot+ softwares. Among the stu died DNA types, salinomycin showed highest affinity to class ic intercalation sites with a free binding energy v alue of -7.33 Kcal/Mol. Salinomycin also demonstrated good affini ty towards potassium channel protein with binding e nergy of - 6.84Kcal/Mol in AutoDock and ACE value of -435.0Kca l/Mol in PatchDock. Finally, among the studied Wnt/ β - catenin signalling proteins, salinomycin showed hig hest affinity towards Protein Kinase A (PKA) with b inding energy of -7.94Kcal/Mol with an inhibition constant of 1.53μ M and towards Casein Kinase 1 gamma (CK1 γ ) with binding energy of -7.81Kcal/Mol with an inhibition constant of 5.45μ M. This molecular docking analysis study concludes that, the mode of action of salinomycin f or its ability to reduce the level of β -catenin and LRP-6 could be due to synergistic inhibition of PKA and CK1 γ . Additionally, if salinomycin is to cause DNA dama ge directly, it would do so at classical intercalation sites. As an ionophore, salinomycin demonstrated good affinity towards human potassium channel. However, the most signific ant report of this study is that, anticancer activi ty of salinomycin by blocking the Wnt/ β -catenin signal could potentially be achieved by in hibiting the activity of phosphorylating proteins PKA and CK1 γ .