The study aimed to compare two insemination strategies in women undergoing in vitro fertilization (IVF) with three to five retrieved oocytes. A total of 462 patients were recruited in our center and obtained three to five oocytes during their first oocyte retrieval between January 2020 and December 2023. Patients whose oocytes underwent overnight insemination were assigned to group A; while those whose oocytes subjected to short-time insemination (4-6h) were recruited as group B. The clinical pregnancy rate, implantation rate, miscarriage rate and multiple pregnancy rates were similar between the two groups (P >0.05). However, group B achieved a higher number of top-quality D3 embryos, a better blastocysts rate, and more frozen embryos compared to group A (P<0.05). The early rescue intracytoplasmic sperm injection (ICSI) rate was significantly higher in group B (6.49% Vs 0.00%, P=0.000), resulting in a notably lower total fertilization failure (TFF) rate in group B (2.16% Vs 7.36%, P=0.009) compared to group A. Short-time insemination strategies significantly increase the rate of available embryos rate and decrease the TFF rate, which may be increases the cumulative live birth rate in IVF treatment. This approach may be particularly suitable for patients with three to five oocytes and a favorable clinical prognosis.