As human umbilical cord mesenchymal stem cells (hUC-MSCs) transplanation may be promising in heart failure treatment, it is important to know whether hypoxic preconditioning (HP) promote hUC-MSCs proliferation and differentiation and protect them against chemical hypoxic damages. This study aimed to investigate the effects of HP on proliferation and differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs). The study also aimed to confirm our hypothesis that HP could promote hUC-MSCs proliferation and differentiation to cardiomyocyte-like cells as well as effectively protecting hUC-MSCs and cardiomyocyte-like cells against chemical hypoxic damages. Isolated hUC-MSCs were cultured in hypoxia at 1%, 3% and 5% O_2 for 72 hours. 5-azacytidine (5-AZA) induced differentiation of hUC-MSCs to cardiomyocyte-like cells was determined by streptavidin-perosidase (SP) immunohistochemical staining and the content of troponin (TnI). Flow cytometry was used to measure cell cycle in hUC-MSCs and cardiomyocyte-like cells. The mitochondrial membrane potential (ΔΨ_m) and mitochondrial Ca^(2+) concentration ([Ca^(2+)]_m), were measured in hUC-MSCs and cardiomyocyte-like cells during chemical hypoxia induced by cobalt chloride (100 μmol/L). HP optimally promoted the proliferation of hUC-MSCs at 3% O_2 and enhanced the differentiation of hUC-MSCs to cardiomyocytelike cells by 5-AZA in a concentration-dependent manner. The cell cycle distribution of cardiomyocytelike cells, but not hUC-MSCs, was clearly changed by HP. Chemical hypoxic damage, decreased ΔΨ_m and increased [Ca^(2+)]_m, were alleviated significantly in HP-treated cells compared with the normaxiatreated cells. The results demonstrate that HP promoted hUC-MSCs proliferation and differentiation to cardiomyocyte-like cells, and protected both cell types against chemical hypoxic damage.