•Estradiol down-regulates the neutrophil influx to the vaginal lumen.•Etradiol down-regulates the gradients of CXCL1.•Estradiol receptor-alpha (ESR1) is essential in the CXCL1 down regulation. Female reproductive mucosa must allow allogenic sperm survival whereas at the same time, avoid pathogen infection. To preserve sperm from neutrophil attack, neutrophils disappear from the vagina during the ovulatory phase (high estradiol); although the mechanisms that regulate neutrophil influx to the vagina during insemination remain controversial. We investigated the sex hormone regulation of the neutrophil migration through the cervix during insemination and revealed that ovulatory estradiol dose fades the CXCL1 epithelial expression in the ectocervix and fornix; hence, retarding neutrophil migration and retaining them in the epithelium. These mechanisms spare sperm from neutrophil attack to preserve reproduction, but might compromise immunity. However, luteal progesterone dose promotes the CXCL1 gradient expression to restore neutrophil migration, to eliminate sperm and prevent sperm associated pathogen dissemination. Surprisingly, these mechanisms are hormone dependent and independent of the insemination. Thus, sex hormones orchestrate tolerance and immunity in the vaginal lumen by regulating neutrophil transepithelial migration in the fornix and ectocervix.