Shewanella oneidensis is a facultative anaerobic γ-proteobacterium possessing remarkably diverse respiratory capacities for reducing various organic and inorganic substrates. As a veteran research model for investigating redox transformations of environmental contaminants the bacterium is well known to be a naturally ampicillin-resistant microorganism. However, in this study we discovered that ampicillin has a significant impact on growth of S. oneidensis. Particularly, cell lysis occurred only with ampicillin at levels ranging from 0.49 to 6.25 µg/ml but not at 50 µg/ml. This phenotype is attributable to insufficient expression of the β-lactamase BlaA. The subsequent analysis revealed that the blaA gene is strongly induced by ampicillin at high (50 µg/ml), but not at low levels (2.5 µg/ml). In addition, we demonstrated that penicillin binding protein 5 (PBP5), the most abundant low molecular weight PBP (LMW PBP), is the only one relevant to β-lactam resistance under the tested conditions. This nonessential PBP, largely resembling its Escherichia coli counterpart in functionality, mediates expression of the blaA gene.