Amniotic mesenchymal stem cells (AMSCs) from livestock are valuable resources for animal reproduction and veterinary therapeutic. The purpose of this study is to explore a suitable way to isolate and culture the buffalo AMSCs (bAMSCs), and to identify their biological characteristics. Digestion with a combination of trypsin-EDTA and collagenase type I could obtain pure bAMSCs more effectively than trypsin-EDTA or collagenase type I alone. bAMSCs could proliferate steadily in vitro culture and exhibited fibroblastic-like morphology in vortex-shaped colony. bAMSCs were positive for MSC-specific markers CD44, CD90, CD105, CD73, β-integrin (CD29) and CD166, and pluripotent markers OCT4, SOX2, NANOG, REX-1, SSEA-1, SSEA-4 and TRA-1-81, but negative for hematopoietic markers CD34, CD45 and epithelial cells specific marker Cytokeratin 18. In addition, bAMSCs were capable of differentiating into adipogenic, osteogenic, chondrogenic and neural lineages, with expression of FABP4, Ost, ACAN, COL2A1, Nestin and β III-tubulin. Glycogen synthase kinase 3 inhibitor: kenpaullone promoted bAMSCs to differentiate into neural lineage. This study provides an effective protocol to obtain and characterize bAMSCs, which have proven useful as a cell resource for buffalo cell reprogramming studies and transgenic animal production.