In Brucella abortus, two small RNAs (sRNAs), AbcR1 and AbcR2, are responsible for regulating transcripts encoding ABC-type transport systems. AbcR1 and AbcR2 are required for Brucella virulence, as a double chromosomal deletion of both sRNAs results in attenuation in mice. Although these sRNAs are responsible for targeting transcripts for degradation, the mechanism utilized by the AbcR sRNAs to regulate mRNA in Brucella has not been described. Here, two motifs (M1 and M2) were identified in AbcR1 and AbcR2, and complementary motif sequences were defined in AbcR-regulated transcripts. Site-directed mutagenesis of M1 or M2 or of both M1 and M2 in the sRNAs revealed transcripts to be targeted by one or both motifs. Electrophoretic mobility shift assays revealed direct, concentration-dependent binding of both AbcR sRNAs to a target mRNA sequence. These experiments genetically and biochemically characterized two indispensable motifs within the AbcR sRNAs that bind to and regulate transcripts. Additionally, cellular and animal models of infection demonstrated that only M2 in the AbcR sRNAs is required for Brucella virulence. Furthermore, one of the M2-regulated targets, BAB2_0612, was found to be critical for the virulence of B. abortus in a mouse model of infection. Although these sRNAs are highly conserved among Alphaproteobacteria, the present report displays how gene regulation mediated by the AbcR sRNAs has diverged to meet the intricate regulatory requirements of each particular organism and its unique biological niche. IMPORTANCE Small RNAs (sRNAs) are important components of bacterial regulation, allowing organisms to quickly adapt to changes in their environments. The AbcR sRNAs are highly conserved throughout the Alphaproteobacteria and negatively regulate myriad transcripts, many encoding ABC-type transport systems. In Brucella abortus, AbcR1 and AbcR2 are functionally redundant, as only a double abcR1 abcR2 (abcR1/2) deletion results in attenuation in vitro and in vivo. In the present study, we confirmed that the AbcR sRNAs have redundant regulatory functions and defined two six-nucleotide motifs, M1 and M2, that the AbcR sRNAs utilize to control gene expression. Importantly, only M2 was linked to B. abortus virulence. Further investigation of M2-regulated targets identified BAB2_0612 as critical for colonization of B. abortus in mice, highlighting the significance of AbcR M2-regulated transcripts for Brucella infection. Overall, our findings define the molecular mechanism of the virulence-associated AbcR system in the pathogenic bacterium B. abortus.