This work aimed to study the beneficial role of human umbilical cord blood-derived mesenchymal stem cell-conditioned medium (MSC-CM) in hypoxia-induced apoptosis in H9c2 cardiomyoblasts, in which the serine/heroine kinases (Akt) pathway would be involved. For this, CM was collected by culturing MSCs in serum-free DMEM medium for 24 h, and paracrine factors were analyzed by protein chip. H9c2 cells were divided into the following groups: control group, hypoxia group, MSC-CM intervention group (CM group), MSC-CM + Akt phosphorylation inhibitor (LY294002) group (LY group). Apoptosis of the H9c2 cells was tested with chromatin dye Hoechst 33342 and FITC-conjugated Annexin V apoptosis detection kit by flow cytometer after a hypoxia/serum deprivation (H/SD) for 24 h. The apoptosis-related proteins were evaluated by Western blot. MSC-CM displayed significantly elevated levels of growth factors, anti-inflammatory, and anti-apoptosis cytokines. On Hoechst 33342 apoptosis staining, the H9c2 cell morphology displayed a lower proportion of apoptosis in the CM group than those in the hypoxia group, while apoptosis was increased in LY group. Flow cytometer analysis revealed the apoptosis ratio in the CM group was lower than the hypoxia group (12.34 +/- 2.00% vs. 21.73 +/- 2.58%; p < 0.05), while the LY group was significantly higher (22.54 +/- 3.89%). Active caspase-3 expression was increased in hypoxia group than control group (p < 0.05), but decreased in CM group (p < 0.01). Umbilical cord blood-derived mesenchymal stem cell-conditioned media secrete multiple paracrine factors that are able to inhibit hypoxia-induced H9c2 cardiomyoblasts apoptosis, and in which the activation of Akt phosphorylation is involved to achieve the protective effect.