Mesenchymal stem cells (MSCs) are extremely valuable in veterinary and human medicine due to their potential application in regenerative medicine. The purpose of this study was to isolate, differentiate and characterize bovine MSCs (bMSCs) from fetal adnexa including amniotic fluid (AF) and Wharton’s jelly (WJ) of Nili-Ravi buffalo during the second trimester. After slaughtering of the animals, pregnant uteri (n=3) were retrieved and properly disinfected before bMSC isolation. The cells from AF were isolated by centrifugation at 400g for 10 minutes, while from WJ by enzymatic digestion with trypsin-EDTA (0.05%). The isolated cells were studied for their plastic adherence, phenotype identification, metabolic activity and in vitro differentiation ability. The isolated AF and WJ bMSCs were fibroblast-like cells in their phenotype, adhered to plastic, showed similar metabolic potential and population doubling time (PDT), but proliferative activity was initially higher (P<0.05) in WJ-bMSCs. When appropriately induced, both cell types showed mesodermal differentiation into adipogenic and osteogenic lineages which was further affirmed by immunolocalization of fatty acid‐binding protein 4 (FABP4) and osteopontin (OST), respectively. However, image analysis revealed that the osteogenic activity of WJ-bMSCs was significantly higher (P<0.05) than that of AF-bMSCs. MSC surface markers (CD73 and CD90) were also positively expressed by both cell types. The study showed that the fetal adnexa of buffalo is a rich source of MSCs for culture and has robust differentiation capabilities.