Objective: The objective of this study was to estimate whether the time interval between two intrauterine inseminations (IUI) treatments needs to be extended by one menstrual cycle or more, and whether this will have an impact on the clinical pregnancy rate (CPR).Study Design: Retrospective cohort study.Study site: The study site was the reproductive medicine center of a teaching hospital.Patient(s): The subjects were women and their husbands who received two or more intrauterine insemination in our reproductive medicine center due to mild infertility in the period from January 2014 to December 2020. Patients were divided into 2 groups according to the number of days between the last menstrual day(LMD)and the previous IUI operation day(POD), continuous group (the time from the LMD to POD ≤ 34 days) and delayed group (the time from the LMD to POD ≥ 35 days). If the previous cycle was a pregnancy or abortion cycle, the next cycle immediately adjacent to it was defined as a new cycle, and the days between the two cycles were not included in the study.Intervention(s):No intervention.Main Outcome Measure(s): A total of 1491 cycles were finally included in the study.990 cycles followed by the second IUI cycle after the previous failure,501 cycles at least one menstrual cycle was separated between two IUI treatments. The primary outcome measure was clinical pregnancy rate (CPR), with secondary outcomes including abortion rate and live birth rate. Differences in clinical pregnancy rate (CPR)、abortion rate and live birth rate were compared between the two groups.Result: No significant differences with regard to baseline demographic and the number of treatment cycles, the duration of infertility, the type of infertility, the mode of treatment, and the cause of infertility were observed between the two groups.There were no statistical differences between the delayed group vs continuous group regarding the clinical pregnancy rate(15.0% vs 13.7%), live birth rate(78.7% vs 74.3%), and abortion rate(17.3% vs 18.4%)(P>0.05).The above factors were included for binary logistic regression analysis. The observed difference in clinical pregnancy rate between the groups was not statistically significant after adjustment(OR = 1.101,95%CI 0.807-1.499, P=0.546).The all cycles were divided into four groups based on female age. results showed that when the female's age was ≤ 25 years old, the pregnancy rate in the continuous group was 16.5%, which was significantly higher than that in the delayed group by 5.8% (difference 0.107, 95% CI 0.016-0.198, P = 0.055), approached, but did not reach, statistical significance. When the female was 30-35 years old, the pregnancy rate in the delayed group was 19.4%, which was significantly higher than 10.9% in the continuous group (difference 0.085, 95% CI 0.016-0.154, P = 0.011). The difference between the two groups was statistically significant. The all cycles were divided into three groups based on years of infertility. Our results show that when the number of years of infertility was≤2 years, the clinical pregnancy rate was 20.7% in the delayed group and 12.5% in the continuous group (difference 0.107, 95% CI 0.150-0.014, P = 0.013), statistical significance was maintained. Based on the number of treatment cycles, it is divided into 2 cycles, 3 cycles, and≥4 cycles. The results showed that when≥4 cycles, the pregnancy rate in the continuous group were 19.4%, which was significantly higher than 6.1% in the delayed group (difference 0.133, 95% CI 0.246-0.020, P = 0.038). Statistical significance was maintained at P < 0.05.Conclusions: Overall, prolonging the interval between two IUI did not significantly improve pregnancy outcomes. Yet, for different age stages, duration of infertility, and the number of treatment cycles, we suggest that more flexible treatment strategies can be tried to improve the clinical pregnancy rate.