ABSTRACTColistin is one of the last-resort antibiotics for treating infections caused by multidrug-resistant (MDR) Gram-negative bacteria. However,mcrgenes conferring resistance to colistin have been widely identified, which is considered a global threat to public health. Here, we investigated the prevalence and characteristics ofmcr-harboringEscherichia colistrains isolated from humans, animals, and foods in China by PCR, antimicrobial susceptibility testing, conjugation experiments, molecular typing, genome sequencing, and bioinformatics analysis. In total, 135mcr-1-harboringE. coliisolates were acquired from 847 samples, and 6 isolates carriedmcr-3. Among them, 131 isolates were MDR bacteria. Sixty-five resistance genes conferring resistance to multiple antimicrobials were identified in 135 isolates. The diverse pulsed-field gel electrophoresis (PFGE) patterns and sequence types (STs) ofmcr-1-carrying isolates demonstrated that clonal dissemination was not the dominant mode ofmcr-1transmission. Seven types of plasmids were able to carrymcr-1in this study, including IncI2, IncX4, IncHI2, p0111, IncY, and two hybrid plasmids. The genetic structures carryingmcr-1of 60 isolates were successfully transferred into the recipient, including 25 IncI2 plasmids, 23 IncX4 plasmids, and an IncHI2 plasmid.mcr-1–pap2was the dominantmcr-1-bearing structure, followed by ISApl1–mcr-1–pap2–ISApl1(Tn6330) and ISApl1–mcr-1–pap2, among 7mcr-1-bearing structures of 135 isolates. In conclusion, IncI2, IncX4, and IncHI2 plasmids were the major vectors spreadingmcr-1from different geographical locations and sources. The prevalence of Tn6330may accelerate the transmission ofmcr-1. Continuous surveillance ofmcr-1and variants in bacteria is vital for evaluating the public health risk posed bymcrgenes.IMPORTANCEThe spread of polymyxin-resistant Enterobacteriaceae poses a significant threat to public health and challenges the therapeutic options for treating infections on a global level. In this study,mcr-1-bearing ST10E. coliwas isolated from pigs, pork, and humans simultaneously, which demonstrated that ST10E. coliwas an important vehicle for the spread ofmcr-1among animals, foods, and humans. The high prevalence ofmcr-1-positiveE. colistrains in pigs and pork and the horizontal transmission ofmcr-1-bearing plasmids in diverseE. colistrains suggest that pigs and pork are important sources ofmcr-1-positive strains in humans and pose a potential threat to public health. Additional research on the prevalence and characteristics ofmcr-1-positiveE. coliis still required to facilitate early warning to improve polymyxin management in hospitals.