Rho‐associated coiled‐coil‐forming protein serine/threonine kinase (ROCK) consisting of two isoforms, ROCK‐I and ROCK‐II, functions downstream of the small GTPase Rho for assembly of actomyosin bundles. To examine the role of ROCK isoforms in vivo, we previously generated and examined mice deficient in each of the two isoforms individually. Here, we further examined the in vivo role of ROCK isoforms by generating mice deficient in both isoforms. Cross‐mating of ROCK‐I^+/−ROCK‐II^+/− double heterozygous mice showed that all of the ROCK‐I^−/−ROCK‐II^−/− homozygous mice die in utero before 9.5 days post‐coitum (dpc) and ROCK‐I^−/−ROCK‐II^+/− homo‐heterozygous or ROCK‐I^+/−ROCK‐II^−/− hetero‐homozygous mice die during a period from 9.5 to 12.5 dpc, whereas mice of other genotypes survive until 12.5 dpc with the expected Mendelian ratio. All of the ROCK‐I^+/−ROCK‐II^−/− or ROCK‐I^−/−ROCK‐II^+/− mice showed impaired body turning and defective vascular remodeling in the yolk sac. Impairment of vascular remodeling was also observed in wild‐type embryos treated ex vivo with a ROCK inhibitor, Y‐27632. These results suggest that ROCK isoforms function redundantly during embryogenesis and play a critical role in vascular development.