Objective To establish an easy and efficient method of the isolation, purification and culture of Hamster primary hepatocytes. Methods Hamster Primary Hepatocytes were isolated using two-step collagenase perfusion in situ and purified by centrifugation with Percoll. The yields and viabilities were measured by standard trypan blue exclusion. Hepatocytes were identified using morphologic observation. Cell function was evaluated by Western blot method. Results Under the optical microscope, the obtained hepatocytes exhibited as irregular polygon with two or more circular nucleuses in each cell. The average viability of hepatocytes was 93.7% ± 2.1%. After incubated with adenosine, intracellular AMPK phosphorylation was significantly increased. Conclusion The method for isolating, purifying and culturing Hamster primary hepatocytes was successfully established.