Culturing human pluripotent stem cells (hPSCs) is notoriously challenging, posing a significant barrier to stem cell-based research. The ROCK1/2 kinase inhibitor Y-27632 is widely used to enhance hPSC survival after thawing and passaging and as an agent for imporving the directed differntiation into mature cell types. However, its application varies across laboratories due to a lack of standardisation. Despite its widespread use, the impact of Y-27632 on the epigenetic landscape of hPSCs—critical for maintaining cellular identity and pluripotency—remains unclear. Here, for the first time we investigated whether Y-27632 influences the epigenome and pluropitent state of both human and mouse stem cells. Firstly, we analyzed total levels of key histone modifications H3K27ac, H3K4me1 (Enhancers), H3K36me3 (Gene bodies), H3K27me3, H3K4me3 (Promoters) involved in gene regulation in hPSCs treated with Y-27632 in the different ways reported in the literature. Notably, we observed a rapid (within 90mins) and specific increase in H3K4me3 levels, a histone mark associated with active transcription. ChIP-seq analysis further revealed that Y-27632 induces a genome-wide increase in H3K4me3 levels, suggesting an unknown role for on-target Y-27632 ROCK1/2 inhibtion and chromatin regulation or an uncharacterised off-target effect of the drug. We next assayed the effect of Y-27632 in spontaneous germ layer differentiation using mouse embryoid bodies and directed differentiation of hPSCs to neural progenitor cells. We found that Y-27632 treatment causes dramatic changes the epigenome important for maintaining pluripotency and differentiation. Taken together, our studies have revealed a previosuly unkown epigenetic consequence of Y-27632 in stem cells, and its missue may lead to epigenetic consequences that underly cellular phenotypes not presently controlled for.