Tissue engineering provides a promising method of introducing functional cardiomyocytes (CMs) to damaged myocardium after myocardial infarction; however, finding a biocompatible construct with the chemical and mechanical properties capable of supporting CM function is challenging. Serum albumin hydrogels are novel autogenic scaffolds with elastic properties that can be tailored to mimic the stiffness of native adult myocardium. We assessed the hypothesis that culturing immature CMs on these serum albumin hydrogels would affect CM gene expression and calcium handling. Neonatal cardiomyocyte (NRVM) viability was maintained for at least 14 days on the hydrogels, with clear sarcomeric striations. Cardiac gene expression was quantified using RT-qPCR and demonstrated an up regulation in many genes of cells cultured on hydrogels compared to glass (e.g. relative expression (log 2-ΔΔCt) of ryanodine receptor 2: glass= -2.3±0.5, hydrogel= -0.3±0.1,p<0.01; connexin 43:glass= -1.7±0.5, hydrogel= 0.3±0.1,p<0.01,n=4-6). Compared to glass, NRVMs on hydrogels have an increased time to peak of the calcium transients measured using Fluo-4AM and field stimulated at 1 Hz (tp glass=38±3 ms, tp hydrogel= 54±2 ms, p<0.01,n=4-6). Compared to glass the hydrogels also have a reduced time 50% decay (t50 glass=108±13 ms, t50 hydrogel=78±6 ms, p<0.05,n=4-6) and 80% decay (t80 glass=217±19 ms, t80 hydrogel= 152±10 ms,p<0.05,n=4-6). Human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) were cultured on the hydrogels for up to 28 days. Calcium handling was faster in the iPSC-CMs cultured on the hydrogels in comparison to glass with a reduced time to peak (tp glass=281±43 ms, tp hydrogel= 186±8 ms, p<0.05, n=4) and time to 50% decay (t50 glass=269±15 ms, t50 hydrogel=204±10 ms,p<0.01,n=4) and 90% decay (t90 glass=535±33 ms, t90 hydrogel=397±19 ms, p<0.01,n=4). The serum albumin hydrogels are compatible with NRVM and iPSC-CM culture for at least 28 days. We demonstrate that the serum albumin hydrogels have significant effects on CM calcium cycling and have the potential for use in myocardial repair. Further study is required to determine the mechanisms involved in calcium handling alterations and then assess this engineered patch in vivo for cardiac repair.