Despite a similar mechanism of action underlying their glucose-lowering effects in type 2 diabetes, dipeptidyl peptidase-4 (DPP-4) inhibitors have diverse molecular structures, raising the prospect of agent-specific, glucose-independent actions. To explore the issue of possible DPP-4 inhibitor cardiac heterogeneity, we perfused different DPP-4 inhibitors to beating mouse hearts ex vivo, at concentrations equivalent to peak plasma levels achieved in humans with standard dosing. We studied male and female mice, young non-diabetic mice, and aged diabetic high fat diet-fed mice and observed that linagliptin enhanced recovery after ischemia-reperfusion, whereas sitagliptin, alogliptin, and saxagliptin did not. DPP-4 transcripts were not detected in adult mouse cardiomyocytes by RNA sequencing and the addition of linagliptin caused ≤0.2% of cardiomyocyte genes to be differentially expressed. In contrast, incubation of C166 endothelial cells with linagliptin induced cell signaling characterized by phosphorylation of Akt and endothelial nitric oxide synthase, whereas the nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine increased serine 16 phosphorylation of the calcium regulatory protein, phospholamban in cardiomyocytes. Furthermore, linagliptin increased cardiomyocyte cGMP when cells were co-cultured with C166 endothelial cells, but not when cardiomyocytes were cultured alone. Thus, at a concentration comparable to that achieved in patients, linagliptin has direct effects on mouse hearts. The effects of linagliptin on cardiomyocytes are likely to be either off-target or indirect, mediated through NO generation by the adjacent cardiac endothelium.