Background: Conception rate is an economically important trait in the dairy industry; however, it has decreased dramatically over recent decades. Conception is a complex process including follicle development, ovulation, fertilization, implantation, and placental differentiation and numerous factors contribute to this event. The present study aims to explore the genetics of conception rate in Holsteins using a genome-wide association study (GWAS). Methods and Findings: Our GWAS for conception rate based on 2,559 Holstein sires found that the conception rate is influenced by a single nucleotide polymorphism GA-266del in the 5’ untranslated region of friend leukaemia integration 1 (FLI1). Cows with higher conception rates carried the GA polymorphism in the FLI1 5’ untranslated region. Luciferase assays and quantitative analysis of allele ratios revealed that FLI1 transcripts with the GA polymorphism were expressed at higher levels than those carrying the deletion polymorphism. FLI1 is a member of the ETS gene family of transcription factors and disruption of FLI1 increased natural killer cell population. High levels of natural killer cells were correlated with spontaneous abortion in human. Cows with the deletion polymorphism released higher levels of perforin, a product of natural killer cells, than did cows with the GA polymorphism. Moreover, cows with the deletion polymorphism have lower successful rate for pregnancy after embryo transfer than cows with the GA polymorphism. Conclusions: These observations suggest that cows carrying the deletion polymorphism in FLI1 might have lower conception rates because of the enhanced perforin production. Thus, this study provides novel insights into the role of FLI1 during reproduction process.