Background: We hypothesized that the maximum residual concentrations of trimethoprim allowed in food (Acceptable Daily Intake – ADI) could select for de novo trimethoprim resistance in Escherichia coli in vivo. Methods: We developed models of chronic Escherichia coli infection in Galleria mellonella and exposed them to sub-ADI doses of trimethoprim via single-dosing regimens. The emergence of trimethoprim resistance was assessed via isolation of the target bacteria in selective agar plates. Species identity was confirmed using MALDI-TOF mass spectrometry, and the antibiotic susceptibility of E. coli to trimethoprim was evaluated using the E-test to determine the minimum inhibitory concentration (MIC). Results: Exposure to as low as one-tenth of the ADI dose of the single-dosing regimen of trimethoprim resulted in the selection of trimethoprim resistance in E. coli. Conclusion: We found that trimethoprim doses ten-fold lower than those classified as acceptable for daily intake could induce resistance to trimethoprim in E. coli. These results suggest that it would be prudent to include the induction of antimicrobial resistance as a significant criterion for determining ADIs in food.