The feasibility of separating intact bacterial cells by high-performance ion exchange chromatography typical for macromolecular study was investigated. A common HPLC system coupled with a light scattering detector was employed; TOYOPEARL SuperQ-650C, a beaded anion exchanger, was used as the column media; bacterial cell samples were prepared by suspending colonies in the equilibrium buffer. Piperazine-hydrochloric acid buffer (0.02 M, pH 7.0) was chosen to be the equilibrium buffer after screening different buffer systems. The absorbed cells were eluted by a linear gradient of NaCl from 0 to 1.00 M in the equilibrium buffer within 30 min, at a flow rate of 1.0 mL/min. A distinctive chromatographic profile with high reproducibility and accuracy was obtained with all of the six kinds of bacteria tested. The eluted fractions for each sample were confirmed by microscopic analysis and physiological or biochemical identification to the bacterial cells applied. More importantly, the eluted cells retained full viability. It is apparent that living bacteria cells exhibited similar behaviors and properties with molecules in anion exchange chromatography, implying that chromatographic methods can become an effective approach in the studies of intact bacterial cells.