The interaction of atorvastatin with calf thymus DNA (CT-DNA) was investigated in vitro under simulated physiological conditions by using absorption and emission spectroscopy, viscosity measurements, gel electrophoresis and molecular docking studies. Analysis of UV–vis absorbance spectra indicates the formation of complex between atorvastatin and CT-DNA, and obtained binding constant (Kb = 8.2×104 L. mol−1) is comparable to groove binder drugs. Slight increase of viscosity of CT-DNA demonstrated the groove binding mode. Hoechst 33,258 and methylene blue (MB) displacement studies further confirmed such mode of atorvastatin interaction. Thermodynamic parameters ΔG, ΔH, and ΔS measurements were taken at different temperatures indicated that hydrophobic forces played main role in the binding process. Molecular docking provided detailed computational interaction of atorvastatin with CT-DNA which proved that atorvastatin binds to the groove of CT-DNA. Cleavage experiments showed that atorvastatin does not induce any cleavage under the experimental setup. Finally, all results indicated that atorvastatin interacts with CT-DNA via groove binding mode.