Abstract Background: Gastric stem cells (GSCs) can be isolated from the mucosa of the stomach. They are multipotent stem cells having the ability to proliferate and differentiate into numerous cell lineages when cultured on suitable media. The ability of the gastric stem cells to proliferate and differentiate gives a hope for their potential use in the future as a line for cell therapy. Aim of Study: This study was carried out to isolate GSCs from the mucosa of the stomach of the adult male albino rats, culture these GSCs in suitable medium to allow for their proliferation and characterization. Material and Methods: The GSCs were isolated from the rat stomach, cultured in a suitable medium for proliferation then characterized and identified morphologically and immu-nohistochemically. Results: The GSCs were isolated from the rats' stomach, proliferate in Fetal bovine serum (FBS) enriched DMEM F-12 media with antibiotics to prevent the bacterial and fungal growth and proliferation. The GSCs were identified morpho-logically by the presence of long cytoplasmic processes and the formation of colonies. They were identified immunohis-tochemically by their positivity for CD44 and Nestin antigens and negativity against CD34 antigen. Conclusion: The GSCs can be easily isolated from the stomach of rats, cultured in DMEM F-12 medium with 10% FBS to proliferate rapidly forming large colonies.