A bioactive compound, BR-1 was isolated from the methanolic extract of the leaves of Basella rubra L. Compound BR-1 was characterized and structurally elucidated using different spectroscopic techniques like UV-vis, FTIR, NMR and MS-ESI-TOFM. The compound was identified as an isovitexin (C-glycosylflavone). Isovitexin exhibited dose-dependent cytotoxicity against human colon cancer (FIT-29) cells with IC50 value of 29.44 mu g/mL. The possible reasons behind the anticancer activity of isovitexin were studied. Morphological observations revealed that isovitexin induces pro-apoptotic changes including warping of cells, clustering, and destructive fragmentation of cancer cells. Further, isovitexin induced the loss of intracellular LDH, loss of membrane integrity and fragmentation of DNA of HT-29 cells. Isovitexin triggered the apoptosis of cancer cells by upregulating the caspase-3 and Box levels with simultaneous down regulation of procaspase-3 and Bc1-2 protein expression. Molecular docking studies revealed that isovitexin bound at the catalytic cleft of Bc1-2 by the way of six H-bonding interactions by accepting electrons from Arg-109, Glu-136, Leu-137, Arg-I46, Ala-149 and Glu-152 and thereby effectively inhibited the Bc1-2. Isovitexin gratifies pharmacokinetic bioavailability parameters as evidenced by Lipinski rule and ADMET profile. The results of the present study support the development of natural bioactive compounds as cancer chemotherapeutics.