ABSTRACT Streptomycin (Sm) is a commonly used antibiotic for its efficacy against diverse bacteria. The plant pathogen Agrobacterium fabrum is a model for studying pathogenesis and interkingdom gene transfer. Streptomycin-resistant variants of A. fabrum are commonly employed in genetic analyses, yet mechanisms of resistance and susceptibility to streptomycin in this organism have not previously been investigated. We observe that resistance to a high concentration of streptomycin arises at high frequency in A. fabrum , and we attribute this trait to the presence of a chromosomal gene ( strB ) encoding a putative aminoglycoside phosphotransferase. We show how strB , along with rpsL (encoding ribosomal protein S12) and rsmG (encoding a 16S rRNA methyltransferase), modulates streptomycin sensitivity in A. fabrum . IMPORTANCE The plant pathogen Agrobacterium fabrum is a widely used model bacterium for studying biofilms, bacterial motility, pathogenesis, and gene transfer from bacteria to plants. Streptomycin (Sm) is an aminoglycoside antibiotic known for its broad efficacy against gram-negative bacteria. A. fabrum exhibits endogenous resistance to somewhat high levels of streptomycin, but the mechanism underlying this resistance has not been elucidated. Here, we demonstrate that this resistance is caused by a chromosomally encoded streptomycin-inactivating enzyme, StrB, that has not been previously characterized in A. fabrum . Furthermore, we show how the genes rsmG , rpsL , and strB jointly modulate streptomycin susceptibility in A. fabrum .