Objective To analyze the expression of Nonmuscle Myosin II (NM II) in pharyngeal arches and the function of NM II on the development of pharyngeal arches in zebrafish embryos. Methods The expression of NM II-B in pharyngeal arches in zebrafish embryos was observed by whole-mount in situ hybridization and cryostat section (n=10). The function of NM II-B on the development of pharyngeal arches in zebrafish embryos was analyzed following the comparison of wild type (n=10) and Blebbistatin-treated (n=10 per concentration) zebrafish embryos by cartilage Alcian blue staining. Results The expression of NM II-B in pharyngeal arches in zebrafish was detected at 72 hours post-fertilization (hpf); the expression of NM II-B in pharyngeal arches increased at 120 hpf. The pharyngeal arches in zebrafish appeared obvious developmental defects, and a few of chondrocytes showed significant morphological changes after Blebbistatin inhibition of NM II-B functions, which was at a dose-dependent manner. Conclusion NM II-B regulates the development of pharyngeal arches through affecting the chondrocytes of pharyngeal arches in zebrafish embryos.