Small molecules that interact with G-quadruplex structures formed by the human telomeric region and stabilize them have the potential to evolve as anticancer therapeutic agents. Herein we report the interaction of a putative anticancer agent from a plant source, chelerythrine, with the human telomeric DNA sequence. It has telomerase inhibitory potential as demonstrated from telomerase repeat amplification assay in cancer cell line extract. We have attributed this to the quadruplex binding potential of the molecule and characterized the molecular details of the interaction by means of optical spectroscopy such as absorbance and circular dichroism and calorimetric techniques such as isothermal titration calorimetry and differential scanning calorimetry. The results show that chelerythrine binds with micromolar dissociation constant and 2:1 binding stoichiometry to the human telomeric DNA sequence. Chelerythrine association stabilizes the G-quadruplex. Nuclear magnetic resonance spectroscopy ((1)H and (31)P) shows that chelerythrine binds to both G-quartet and phosphate backbone of the quadruplex leading to quadruplex aggregation. Molecular dynamics simulation studies support the above inferences and provide further insight into the mechanism of ligand binding. The specificity toward quartet binding for chelerythrine is higher compared to that of groove binding. MM-PBSA calculation mines out the energy penalty for quartet binding to be -4.7 kcal/mol, whereas that of the groove binding is -1.7 kcal/mol. We propose that the first chelerythrine molecule binds to the quartet followed by a second molecule which binds to the groove. This second molecule might bring about aggregation of the quadruplex structure which is evident from the results of nuclear magnetic resonance.