To investigate the mechanism of genotype differences in ciprofloxacin (CIP) accumulation, this study was designed to compare the tolerance and metabolic responses to CIP exposure between low (Cutai) and high (Sijiu) CIP-accumulation cultivars of Brassica parachinensis. Decreases in biomass and chlorophyll content were significantly greater (p<0.05) and toxicities were more severe within cell ultrastructures of Cutai compared to Sijiu. A sequential growth test also revealed that Sijiu was more tolerant to CIP stress compared to Cutai. Meanwhile, significantly higher (p<0.05) root parameters and higher areas of the stele and xylem may be responsible for the increased uptake and transport of CIP in Sijiu. Ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC–ESI-MS/MS) analysis revealed that CIP was metabolized to three major metabolites by the hydroxylation and breakdown of the piperazinyl substituent in the CIP molecule. The enhanced metabolic transformation of CIP in Sijiu indicated a more efficient capacity to detoxify, which in turn favored an increased accumulation of CIP in this cultivar. Thus, the present study demonstrated that the stronger tolerance and metabolism of Sijiu to CIP were responsible for its high CIP accumulation, suggesting an evolutionary mechanism for adaptation to environmental stress.