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Objective The aim of this retrospective cohort study was to evaluate the clinical and neonatal outcomes between day 4 and day 5 embryo transfer across different fertilization methods. Methods This retrospective cohort study was conducted between January 2018 and March 2023, enrolling a total of 1,245 fresh embryo transfer cycles, including 1,023 in vitro fertilization (IVF) cycles and 222 intracytoplasmic sperm injection (ICSI) cycles. Among these, IVF cycles included 793 day 4 transfers and 230 day 5 transfers; ICSI cycles included 179 day 4 transfers and 43 day 5 transfers. The study conducted a comparative analysis of clinical pregnancy outcomes between day 4 and day 5 transfers. Results In IVF or ICSI cycles, no significant differences were found in clinical pregnancy rate (CPR), implantation rate (IR), live birth rate (LBR), or other clinical outcomes between transfers of day 4 and day 5 embryos ( P > 0.05). In IVF cycles with single high-quality embryo transfer, the CPR (62.72%, P = 0.026), gestational week of delivery (39 weeks, P = 0.026) of day 4 were significantly higher than day 5 (38 weeks). After controlling for potential confounding factors, the CPR of the day 4 group was also higher than day 5 (OR 0.578, 95% CI 0.352-0.949, P = 0.030). In IVF or ICSI cycles with day 4 high-quality embryo transfer, the LBR (63.06%, P = 0.006; 70.37%, P = 0.006) and multiple pregnancy rate (MPR; 53.29%, P < 0.001; 50%, P < 0.001) of transferring double high-quality embryo were significantly higher than transferring single high-quality embryo (LBR: 50.87%, 44.23%; MPR: 0%, 3.23%). Conclusion In IVF or ICSI cycles, day 4 embryo transfer is considered a viable option or alternative to day 5 blastocyst transfer with no difference in clinical and neonatal outcomes. In IVF cycles with single high embryo transfer, day 4 transfer is recommended due to its significantly higher pregnancy rate compared to day 5 transfer. To reducing MPRs and preterm birth rates (PBRs), day 4 single embryo transfer is recommended if embryos achieve high-quality grade (full compaction) on day 4.