Aims: Cell therapy is a promising treatment for myocardial infarction. Increasingly, cells are being modified to enhance secretion of cardioprotective factors to improve efficacy in vivo. This study utilized human mesenchymal stem cells (MSCs) that were immortalized and engineered to secrete a fusion protein of Glucagon-like Peptide-1 (GLP-1). We have previously shown a cardioprotective effect of this therapy in vivo (1). To investigate direct paracrine effects on apoptosis, these cells were tested against ischemic adult human cardiomyocytes (hCMs) in vitro. Methods and Results: hCMs underwent ischemia for 1 hour before incubation with either: MSC conditioned media, MSC+GLP-1 conditioned media, 100nM GLP-1, 100nM Exendin-4 or normal media. Cells were pre-incubated with the inhibitors Exendin-9, Wortmannin or U0126 to investigate pathways involved. After 24 hours, apoptosis was measured using TUNEL staining (n=8). After 48 hours, cell viability was measured using trypan blue to identify live/dead cells (n=4). MSC+GLP-1 conditioned media significantly reduced TUNEL+ cells compared to media alone (media: 6.14±0.48%, GLP-1: 3.96±0.56%; Ex-4: 5.1±1.22%; MSC media: 3.71±0.56%; MSC+GLP-1 media: 2.97±0.4%, P<0.05). All agonists reduced the number of dead cells (trypan blue stained) compared to normal media (media: 19.05±6.80%; GLP-1: 5.05±0.54%; Ex-4: 7.88±0.39%; MSC media: 5.41±0.39%; MSC+GLP-1 media; 2.58±1.65%, P<0.05). All agonists increased cell viability, normalised to media alone (GLP-1: 8.40±5.77%; Ex-4: 25.48±6.85%; MSC: 26.29±14.21%; MSC+GLP-1: 26.95±7.14%, P=NS). All antagonists/inhibitors appeared to partially block the effects on apoptosis and cell viability. Conclusions: MSC+GLP-1 conditioned media had a significant beneficial effect on both apoptosis and viability of ischemic hCMs in vitro, greater than MSC conditioned media. This effect appeared to be mediated through the GLP-1R and the PI3K and ERK1/2 pathways.