Trifluoroselenomethionine (TFSeM), a novel non-natural amino acid, was synthesized in seven steps from N-(tert-butoxycarbonyl)-L-aspartic acid tert-butyl ester. TFSeM shows enhanced methioninase-induced cytotoxicity toward human colon cancer derived HCT-116 cells compared to selenomethionine (SeM). Mechanistic explanations for this enhanced activity are computationally and experimentally examined. Comparison of TFSeM and SeM by selenium EXAFS and DFT calculations showed them to be spectroscopically and structurally very similar. None-the-less, when two different variants of the protein GB1 were expressed in an E. coli methionine auxotroph cell line using TFSeM and methionine (Met) in a 9:1 molar ratio, it was found that, surprisingly, 85% of the proteins were composed of SeM, even though no SeM had been added, implying loss of the trifluoromethyl group from TFSeM. The recycling of TFSeM to SeM is enzymatically catalyzed by E. coli extracts. However, TFSeM is not a substrate of E. coli methionine adenosyltransferase.