Lactoferrin plays an important role in the innate immune system, with well-characterized antibacterial, antiviral, and immune modulatory properties. The objective of this study was to determine the allele and haplotype frequency of polymorphisms at positions -586, -190, and -28 of the bovine lactoferrin promoter in Holstein-Friesians and to quantify their association with performance using phenotypic data on progeny from 848 sires. Associations between genotypes and performance were quantified using weighted mixed models with genotyped individuals included as a random effect, and average expected relationships among individuals accounted for through a numerator relationship matrix. The dependent variables were daughter yield deviation for production traits and deregressed predicted transmitting ability for calving interval and functional survival. The C to T polymorphism at -586, which distorts a putative activating protein 2 (AP-2) binding site, was associated with a shorter calving interval and higher somatic cell score. The G to A polymorphism at -190, located in a putative selective promoter factor 1 (SP-1) binding site, was associated with a longer calving interval and decreased functional survival. A third polymorphism (A to C) at position -28, found within the noncanonical TATA box, had a tendency to associate with functional survival. On the basis of the data we proposed a haplotype combination that was associated with improved reproductive performance in the Holstein-Friesian breed. We hypothesized that the observable phenotypic associations with lactoferrin promoter polymorphisms can potentially be explained by allele-specific differences in constitutive or inducible levels of gene expression. The lack of a pleiotropic effect of the single nucleotide polymorphisms studied on both fertility and milk production traits strengthens the importance of these polymorphisms, or at least the lactoferrin promoter, in selection for improved fertility.