To investigate the occurrence and development state of embryo vacuoles between the 8-cell and morula stages, and to explore how vacuoles affected the development of embryos. A retrospective study of a cohort of 422 patients undergoing conventional in vitro fertilization or intracytoplasmic sperm injection. With the help of time-lapse imaging, the development processes and outcomes of good quality embryos with or without vacuoles were analyzed. Vacuole positive embryos had significantly lower blastulation rate and good quality blastulation rate than vacuole negative embryos, p < 0.05. Compared to vacuole negative embryos, the number of best and good quality blastocysts was significantly reduced, while the number of fair and discarded ones was significantly increased, p < 0.05. The average starting time of vacuolization was 73.7 ± 9.3 h after insemination. The proportion of blastomeres affected by vacuoles was associated with embryonic developmental potential. Vacuolization on Day 3 and Day 4 was frequently observed and was detrimental to embryo development. The proportion of blastomeres affected by vacuoles may be an indicator of embryo developmental potential.