A new mobile colistin resistance gene, mcr-3, has been discovered in E. coli of pig origin. The novel mcr-3 gene was discovered when a colistin-resistant Escherichia coli isolate tested negative for both mcr-1 and mcr-2. This novel mobile colistin resistance gene may already be widely disseminated. Screening for the mcr-3 gene should be urgently included in the surveillance of colistin-resistant Gram-negative pathogens from animals, humans, and the environment.