Loss of c-JUN leads to early mouse embryonic death, possibly because of a failure to develop a normal cardiac system. How c-JUN regulates human cardiomyocyte cell fate remains unknown. Here, we used the in vitro differentiation of human pluripotent stem cells into cardiomyocytes to study the role of c-JUN. Surprisingly, the knockout of c-JUN improved cardiomyocyte generation, as determined by the number of TNNT2+ cells. ATAC-seq data showed that the c-JUN defect led to increased chromatin accessibility on critical regulatory elements related to cardiomyocyte development. ChIP-seq data showed that the knockout c-JUN increased RBBP5 and SETD1B expression, leading to improved H3K4me3 deposition on key genes that regulate cardiogenesis. The c-JUN KO phenotype could be copied using the histone demethylase inhibitor CPI-455, which also up-regulated H3K4me3 levels and increased cardiomyocyte generation. Single-cell RNA-seq data defined three cell branches, and knockout c-JUN activated more regulons that are related to cardiogenesis. In summary, our data demonstrated that c-JUN could regulate cardiomyocyte cell fate by modulating H3K4me3 modification and chromatin accessibility and shed light on how c-JUN regulates heart development in humans.© 2023 Zhong et al.