Tobacco smoking was one of the important adverse factors for congenital heart disease. The effects of nicotine, the main component of tobacco, on human embryonic cardiogenesis and related mechanisms remain poorly understood. This work used single-cell RNA sequencing to investigate the effects of nicotine on human embryonic stem cell (hESC) line H9 and its underlying mechanisms during cardiac differentiation. H9 was cultured in feeder-free medium and differentiated in cardiac condition medium when cells reached 90% confluent. Cell viability was detected by MTT after different concentration of nicotine treatment. Different expressed genes during cardiac differentiation was analyzed by single-cell RNA sequencing (scRNA-seq). Key gene expressions were confirmed by qPCR and Western blot. Results showed that 0.1μM-10μM nicotine did not affect H9 cell proliferation. Nicotine 1 μM down-regulated cardiac progenitor cell, mesoderm cell, smooth muscle cell and neural crest cell relatively. Snail1/2 regulating endocardial cushion development were downregulated apparently at differention day 6. Nicotine didn't affect bry-1 and mesp-1 but inhibited cardiac transcript factors. Consequently, the expression of cTnI, a marker of cardiomyocytes was decreased significantly. The data suggest direct adverse effects of nicotine on heart development at the single-cell level and offer a new approach for estimate drug and environmental toxicity on the pathogenesis of the embryonic cardiovascular system development. Keywords: Cardiogenic differentiation; Embryonic stem cell; Single-cell RNA sequencing; nicotine.