Multipotent perivascular cells (PVCs) have recently gained attention as an alternative source for cell-based regenerative medicine. Because of their rarity in human tissues, the development of efficient methods to isolate and expand PVCs from various fetal and adult tissues is necessary to obtain a clinically relevant number of cells that maintain progenitor potency. We report a simple non-enzymatic isolation (NE) method of PVCs from human umbilical cord (HUC) and compare its efficiency with the conventional collagenase treatment method (CT) in terms of proliferation, immunophenotype, clonogenic capacity, and differentiation potential. Cells isolated by NE expressed the accepted surface marker profile of PVCs and possessed multilineage differentiation potential. Whereas both methods provided similar patterns or levels of immunophenotypes and proliferation, PVCs obtained by NE maintained a higher level of CD146(+) frequency compared with that of CT over passages and displayed greater in vitro osteogenic differentiation potential and clonogenic capacity than CT-PVCs. We assess the potential of various exogenous factors to boost the proliferation of NE- and CT-PVCs in vitro. Supplementation of basic fibroblast growth factor (bFGF) provided optimal conditions that significantly enhanced their proliferation rate. This treatment drove the cells into S phase and increased the proportion of stage-specific antigen-4-positive population without altering other immunophenotypes. Thus, the NE method with bFGF supplementation offers an alternative way for obtaining sufficient numbers of HUCPVCs that have good clonogenic and differentiation potential and that are applicable at therapeutic doses for regenerative medicine.