The interaction between prodigiosin (PG) and calf thymus DNA (ct-DNA) was investigated firstly by using UV–Visible (UV–Vis), fluorescence, Fourier transform infrared (FT-IR), circular dichroism (CD) spectroscopies and viscosity measurement in Tris–HCl buffer solution (pH 6.8). The experimental results indicated that PG intercalated into the DNA helix. Upon addition of ct-DNA, PG showed hypochromic effect and slight redshift in the absorption spectra, and the melting temperature of ct-DNA was increased by from 58 to 64°C. Furthermore, FT-IR spectrum and CD spectra also suggested that the partial bases of ct-DNA react with prodigiosin. The fluorescence quenching mechanism was studied using ethidium bromide as a DNA probe, The binding constants of PG with ct-DNA in the presence of EB are 4.46×10 4 and 2.32×10 4 M −1 at 298 and 310K, respectively, and the corresponding thermodynamic parameters ΔG, ΔH, ΔS at various temperatures were obtained.