The NRAMP1 gene encodes a divalent cation transporter, located in the phagolysosomal membrane of macrophages, that has been associated with resistance to intracellular pathogens. In cattle, natural resistance against brucellosis has been associated with polymorphisms at the 3' untranslated region (3'UTR) of the NRAMP1 gene, which are detectable by single-strand conformational analysis (SSCA). This study aimed to evaluate the association between NRAMP1 3'UTR polymorphisms and resistance against bovine brucellosis in experimental and natural infections. In experimentally infected pregnant cows, abortion occurred in 42.1% of cows with a resistant genotype (SSCA(r); n = 19) and in 43.1% of those with a susceptible genotype (SSCA(s); n = 23). Furthermore, no association between intensity of pathological changes and genotype was detected. In a farm with a very high prevalence of bovine brucellosis, the percentages of strains of the SSCA(r) genotype were 86 and 84% in serologically positive (n = 64) and negative (n = 36) cows, respectively. Therefore, no association was found between the NRAMP1-resistant allele and the resistant phenotype in either experimental or naturally occurring brucellosis. To further support these results, bacterial intracellular survival was assessed in bovine monocyte-derived macrophages from cattle with either the resistant or susceptible genotype. In agreement with our previous results, no difference was observed in the rates of intracellular survival of B. abortus within macrophages from cattle with susceptible or resistant genotypes. Taken together, these results indicate that these polymorphisms at the NRAMP1 3'UTR do not affect resistance against B. abortus in cattle and that they are therefore not suitable markers of natural resistance against bovine brucellosis.