Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) play a critical role in most translational and clinical applications. Although glucose starvation (GS) has been evaluated during cellular purification, there has been no comprehensive evaluation of the transcriptional heterogeneity of these cells. Here, we applied GS for 3 days starting at day 10 of differentiation, and then, harvested hiPSC-CMs at day 20 for single-cell RNA sequencing (scRNA-seq). We found that GS dramatically reduced the proportion of non-cardiomyocytes cells and increased the number of late-stage cardiomyocytes. We also recorded an increase in the expression of MYH6, MYH7, ACTN2, TNNT2, and several other genes associated with the structural and functional maturation of cardiomyocytes. Further analysis indicated that these changes were focused on the signaling pathways involved in the regulation of the actin cytoskeleton, cardiac muscle development, and cardiac muscle contraction. Finally, pseudotime analysis revealed that GS hiPSC-CMs developed in a more mature direction. Together, these results suggest that GS treatment improves the purity and maturation of hiPSC-CMs, which should increase the feasibility of hiPSC-CMs applications. (C) 2020 Elsevier Inc. All rights reserved.