Plasmid-mediated quinolone resistance due to Qnr-like proteins is increasingly reported worldwide. While the origin of qnrA is Shewanella algae, the reservoirs of qnrB and qnrS remain unknown, but they were thought to be related to Vibrionaceae species with chromosome-encoded Qnr-like determinants. In this study, in silico analysis of Vibrio splendidus strain 12B01 genome identified an open reading frame (V12B01_17906) encoding a 218-amino-acid protein sharing 84% and 88% identity with QnrS1 and QnrS2, respectively. The qnrVS1 and qnrVS2 genes were PCR-amplified from V. splendidus strains 12B01 (seawater, US) and LGP32 (oyster, France), cloned into pCR-BluntII-TOPO, and overexpressed in Escherichia coli TOP10. Recombinant plasmids pVS1 (qnrVS1) and pVS2 (qnrVS2) conferred an 8-fold increase in nalidixic acid MIC and 4-16-fold increases in fluoroquinolone MICs compared to E. coli TOP10, similar to pS1 (qnrS1) and pS2 (qnrS2). QnrVS1 and QnrVS2 shared 98.1% identity, high identity with QnrS1/QnrS2, and their GC contents (45.2%, 45.1%) were close to those of qnrS1/qnrS2 (43.8%). These results suggest that gram-negative bacteria in aquatic environments may be reservoirs of plasmid-mediated Qnr-like determinants, and the plasmid-encoded QnrS determinant is likely closely related to V. splendidus.