Purpose The culture medium plays an important role in embryonic development and subsequent pregnancy outcomes of in vitro fertilization (IVF) cycles. The sequential culture media of Vitrolife and Cook are the two most commonly used reagents in China. This study aimed to assess their effects on IVF success rates, obstetric outcomes and neonatal outcomes. Methods This was a retrospective cohort study on 6352 patients undergoing first IVF attempts between January 2018 and December 2019, resulting in 3153 clinical pregnancies, 2646 live births, and 2668 babies. Patients were grouped according to the culture media they used. There were 4680 patients in the Vitrolife group and 1672 patients in the Cook group. The primary outcome measures were maternal and neonatal outcomes. ANOVA and Chi-square tests were used for statistical comparison, and multivariate logistic regression and multivariate general linear model were used to adjust for potential confounders. Results The rates of live birth, clinical pregnancy, monozygotic twins, miscarriage, and ectopic pregnancy were all comparable between the groups of Vitrolife and Cook. The incidence of placenta previa was higher in the Vitrolife group [4.86 vs. 3.09%, adjusted odds ratio = 2.048 (1.146-3.657)]. The incidence of macrosomia was higher in the Cook group [7.51 vs. 5.39%, adjusted odds ratio = 1.445 (1.010-2.069)]. Conclusion The culture media of Vitrolife and Cook are comparably effective in IVF success. Vitrolife is associated with a higher risk of placenta previa, while Cook is associated with a higher risk of macrosomia.