Rho-associated protein kinase (ROCK) inhibitor, Y-27632, is an indispensable chemical molecule to maintain the viability of single-dissociated human induced pluripotent stem cells (hiPSCs) and to form cell aggregates in floating cultures. In this study, we investigated the effect of Y-27632 on the cardiac differentiation of the cell aggregates of hiPS cell line, 201B7. When Y-27632 was not added to the floating culture, the dissociated hiPSCs died and no cell aggregates were formed. The presence of greater than 10 μM Y-27632 was required to form spherical cell aggregates from the dissociated hiPSCs. However, Y-27632 used in the floating culture of the dissociated hiPSCs to form cell aggregates exhibited an inhibitory effect on cardiac differentiation in the adherent culture of cell aggregates. When 30 μM Y-27632 was added to the floating cultures, the extensibility of outgrowth from the cell aggregates was relatively lowered, and the initial time of contraction (the generation of beating cardiomyocytes) was markedly delayed in the adherent cultures. Moreover, the expression levels of the early cardiac differentiation-related genes of NKX2.5 and TNNT2 were decreased with increasing Y-27632 concentration. These results indicate that Y-27632 applied to the floating cultures for cell aggregate formation adversely affected the early cardiac differentiation in the following adherent cultures, although there was no influence on the final cardiac differentiation levels.