This study assessed first the impact of endometrial Tiam1/Rac1 signals and microRNA-22 (miR-22) on embryo implantation in mice, and then the expression of the above three genes in the endometrium during the embryo implantation window in the natural menstrual cycle in women with repeated implantation failure (RIF) after in vitro fertilization treatment. Four hundred fifty-two Kun-ming female mice and 200 women (70 infertility patients with RIF, 130 women as controls) were entered into this study. Endometrial Tiam1/Rac1 signals and miR-22 expression were studied in clinical and mouse samples and serum estrogen (E₂) and progesterone (P) were analyzed in clinical subjects. A pregnant mouse model based on an endometrial miR-22 and Tiam1 mRNA expression trend of patients with RIF was constructed and then the embryo implantation numbers were analyzed, and an ovariectomized mouse model was used to assess correlations of expression of these three genes with E₂ and P. The results showed that during the embryo implantation window in the natural menstrual cycle, endometrial miR-22 was significantly higher whereas Tiam1/Rac1 signals were notably lower in patients with RIF than in controls, and the P:E₂ ratio was statistically lower in the RIF group. Tiam1/Rac1 signal down-regulation and miR-22 up-regulation contributed to the inhibition of embryo implantation in mice. We also found a suppressive effect of miR-22 up-regulation on Tiam1/Rac1 signal expression, and reciprocal regulation of E₂ and P for these three genes' expression in mice. In conclusion, miR-22 up-regulation and Tiam1/Rac1 signal down-regulation inhibited embryo implantation in mice; this mechanism may be partially due to the suppressive effect of miR-22 on Tiam1 expression, and is regulated to some extent by serum E₂ and P. Our findings provide evidence that endometrial Tiam1/Rac1 signal down-regulation along with miR-22 up-regulation during embryo implantation window in the natural menstrual cycle may be one of the reasons for the failure of embryo implantation in patients with RIF.