OBJECTIVE We investigated the effect of insemination timing on pregnancy outcomes in intrauterine insemination (IUI) cycles. METHODS This is a retrospective study of 411 IUI cycles performed with a diagnosis of unexplained infertility and male factor infertility. The cycles were divided according to the interval between insemination and ovulation: ≤36 h, 36-37 h, 37-38 h and >38 h. The overall pregnancy rate, chemical pregnancy rate and clinical pregnancy rate were compared. We also analyzed the association between pregnancy outcomes and clinical characteristics, including age, duration of infertility, sperm concentration, body mass index (BMI), anti-Müllerian hormone (AMH) and number of mature follicles at ovulation. RESULTS There were no differences regarding age, duration of infertility, BMI, AMH, sperm concentration and number of mature follicles between different IUI timing groups. Sperm morphology was significantly lower in ≤36 h group (5.3 ± 1.4) compared to 36-37 h, 37-38 h and >38 h (6.3 ± 2.5 vs 6.5 ± 2.7 vs 6.5 ± 3.5, P = 0.004) groups. The ≤36 h group showed lowest total pregnancy rate (5.0%) compared to other IUI timings (21.8% vs 24.8% vs 20.0%, P = 0.05). Multivariate analysis showed that sperm morphology was associated with pregnancy in 36-37 h (odd ratio 1.42, 95% confidence interval 1.03-1.95, P = 0.02). CONCLUSIONS Insemination at least 36 h after ovulation is associated with increased pregnancy rate compared to IUIs performed ≤36 h following ovulation.