Cardiomyocytes (CMs) are non-regenerative. Self-renewable pluripotent human embryonic stem cells (hESCs) can differentiate into CMs for cell-based therapies. We recently reported that Ca²⁺ handling, crucial to excitation-contraction coupling, of hESC-derived CMs (hESC-CMs) is functional but immature. Such immature properties as smaller cytosolic Ca²⁺ transient amplitudes, slower kinetics and reduced Ca²⁺ content of sarcoplasmic reticulum (SR) can be attributed to the differential developmental expression profiles of specific Ca²⁺-handling and regulatory proteins in hESC-CMs and their adult counterparts. In particular, calsequestrin (CSQ), the most abundant, high-capacity but low-affinity Ca²⁺-binding protein in the SR that is anchored to the ryanodine receptor, is robustly expressed in adult CMs BUT completely absent in hESC-CMs. Here we hypothesized that gene transfer of CSQ in hESC-CMs suffices to induce functional improvement of SR. Transduction of hESC-CMs by the recombinant adenovirus Ad-CMV-CSQ-IRES-GFP (Ad-CSQ) significantly increased the transient amplitude, upstroke velocity, and transient decay in comparison to the control Ad-CMV-GFP (Ad-GFP) and Ad-CMV-CSQ-IRES-GFP (Ad-CSQ, which mediated the expression of a non-functional, truncated version of CSQ) groups. Ad-CSQ increased the SR Ca²⁺ content but did not alter L-type Ca²⁺ current. Pharmacologically, untransduced wildtype, Ad-GFP-, Ad-CSQ- and Ad-CSQ-transduced hESC-CMs behaved similarly. While ryanodine significantly reduced the Ca²⁺ transient amplitude and slowed the upstroke, thapsigargin slowed the decay. Neither triadin nor junctin was affected. We conclude that CSQ expression in hESC-CMs facilitates Ca²⁺ handling maturation. Our results shed insights into the suitability of hESC-CMs for therapies and as certain heart disease models for drug screening.