The binding characteristics of sodium saccharin (SSA), an artificial sweetener, with calf thymus DNA (ctDNA) were investigated by multispectroscopic techniques, chemometrics, and molecular simulation. A combined fluorescence and UV-vis spectroscopic data matrix was resolved by the multivariate curve resolution-alternating least-squares (MCR-ALS) chemometrics algorithm. The MCR-ALS analysis extracted simultaneously the concentration profiles and spectra for the three components (SSA, ctDNA, and SSA-ctDNA complex) to quantitatively monitor the SSA-ctDNA interaction, which is difficult to perform by conventional spectroscopic approach. The binding mode of SSA to ctDNA was principally through groove binding as revealed by ctDNA melting temperature studies, viscosity measurements, and iodide and salt quenching effects. Analysis of the Fourier transform infrared and circular dichroism spectra as well as molecular docking indicated that SSA preferentially bound to the guanine base of ctDNA and led to a transformation from B-like DNA structure to A-like conformation. Moreover, gel electrophoresis results suggested that SSA did not induce any significant cleavage in plasmid DNA.