Colicin V (ColV) is a peptide antibiotic that kills sensitive cells by disrupting their membrane potential once it gains access to the inner membrane from the periplasmic face. Recently, we constructed a translocation suicide probe, RR-ColV, that is translocated into the periplasm via the TAT pathway and thus kills the host cells. In this study, we obtained an RR-ColV-resistant mutant by using random Tn10 transposition mutagenesis. Sequencing analysis revealed that the mutant carried a Tn10 insertion in the sdaC (also called dcrA) gene, which is involved in serine uptake and is required for C1 phage adsorption. ColV activity was detected both in the cytoplasm and in the periplasm of this mutant, indicating that RR-ColV was translocated into the periplasm but failed to interact with the inner membrane. The sdaC::Tn10 mutant was resistant only to ColV and remained sensitive to colicins Ia, E3, and A. Most importantly, the sdaC::Tn10 mutant was killed when ColV was anchored to the periplasmic face of the inner membrane by fusion to EtpM, a type II integral membrane protein. Taken together, these results suggest that the SdaC/DcrA protein serves as a specific inner membrane receptor for ColV.