Rho-associated kinase (ROCK) is a serine/threonine kinase working in the Rho signaling to actin cytoskeleton. We previously reported that loss of ROCK-I results in the eyelid open at birth (EOB) and omphalocele phenotype in mice, while loss of ROCK-II results in placental dysfunction leading to intrauterine growth retardation and fetal death. Here, we report that after backcross to the C57BL/6 N genetic background, ROCK-II knockout (KO) neonates are born also with open eyelid and umbilical hernia, a phenotype similar to that of ROCK-I KO mice. ROCK-II KO embryos show impaired extension of the eyelid epithelial sheet with disorganized actin bundles in the leading edge of the sheet. These results suggest that ROCK-I and ROCK-II cooperatively regulates the assembly of actin bundles essential for closure of the eyelid and ventral body wall in mouse embryos. Consistently, ROCK-I+/–ROCK-II+/– double heterozygous mice also show the EOB and omphalocele phenotype.