DNA conformational change and breakage induced by Cr(VI)-GSH interaction were characterized by the integrated tools of electrochemistry, atomic force microscopy (AFM), and DNA electrophoresis. While electrochemistry confirmed the formation of the active species generated from Cr(VI)-GSH reduction, which causes the DNA conformational changes, AFM imaging vividly demonstrated the stepwise process of the DNA denaturation and breakage for the first time. Our DNA electrophoresis further validated that the DNA breakage occurs unevenly at both of the single strands of the molecule. A scheme was drawn based on the experimental observations to explain the phenomenon of the Cr-induced DNA cleavage.