Aims:  The aims of this study were to identify analogues of L‐proline which inhibit the growth of Escherichia coli in both laboratory culture media and normal human urine and to study their mechanisms of uptake. Methods and Results:  The susceptibility of E. coli to L‐proline analogues was studied by radial streak assays on agar plates and by minimal inhibitory concentration determinations in liquid media. Only L‐selenaproline (SCA) inhibited growth in Mueller–Hinton medium and human urine as well as in glucose minimal medium. L‐Proline did not prevent the inhibition of growth by SCA and strains defective in L‐proline transport were as susceptible to SCA as wild‐type strains. However, E. coli was resistant to SCA in the presence of L‐cysteine and L‐cystine. Spontaneous mutants selected for resistance to SCA or L‐selenocystine were resistant to the other compound and had reduced growth in minimal medium containing L‐cysteine or L‐cystine as the sole sulfur source. Conclusions:  L‐selenaproline inhibited the growth of E. coli under conditions that may occur in the urinary tract and appeared to be taken up by the L‐cystine transport system. Significance and Impact of the Study:  Although urinary tract infections caused by E. coli can be treated with sulfamethoxazole/trimethoprim and quinolones, resistance to these antibiotics has been increasing. These results suggest that L‐selenaproline may represent a new class of compounds that could be used to treat these infections.