The introduction and wide use of antibacterial drugs has resulted in the emergence of resistant organisms. DfrB dihydrofolate reductase (DHFR) is a bacterial enzyme that is uniquely associated with mobile gene cassettes within integrons, and confers resistance to the drug trimethoprim. This enzyme has intrigued microbiologists since it was discovered more than thirty years ago because of its simple structure, enzymatic inefficiency and its virtual insensitivity to trimethoprim. Here, for the first time, a comprehensive discussion of genetic, evolutionary, structural and functional studies of this enzyme is presented together. This information supports the ideas that DfrB DHFR is a poorly adapted catalyst and has recently been recruited to perform a novel enzymatic activity in response to selective pressure.