Cleft palate is among the most common birth defects with an impact on swallowing and speaking and is difficult to diagnose with ultrasound during pregnancy. In this study, we systematically capture the cellular composition of all-trans retinoic acid (atRA)-exposed and normal embryonic gestation 16.5 days mouse palate by the single-cell RNA sequencing technique. The authors identified 14 major cell types with the largest proportion of fibroblasts. The proportion of myeloid cells in atRA-exposed palate was markedly higher than those in the normal palate tissue, especially M1-like macrophages and monocytes. The upregulated genes of the different expression genes between atRA-exposed palate and normal palate tissue were linked to the biological processes of leukocyte chemotaxis and migration. Protein TLR2, CXCR4, THBS1, MRC1, transcription factor encoding genes Cebpb, Fos, Jun, Rela, and signaling pathway IL-17 and phagosome were found to be significantly involved in these processes. Subsequently, cellular communication network analysis suggested that myeloid-centered cell interactions SELL, SELPLG, MIF, CXCL, ANNEXIN, THBS, and NECTIN were significantly more activated in atRA-exposed palate. Overall, we delineate the single-cell landscape of atRA-induced cleft palate, revealing the effects of overexposure to atRA during palate tissue development and providing insights for the diagnosis of cleft palate.