T cells recognise and react to the presentation of foreign antigens displayed on the surface of all nucleated human cells. Peptide and lipid antigen presentation has dominated immunological research in this domain, but the lesser known MR1-mediated metabolite presentation system is emerging as an intriguing potential therapeutic target. MR1 was originally shown to present microbially derived vitamin B2 precursors to signal a localised infection to specialised T-cells, but evolutionary conservation of the MR1 gene indicates a broader purpose, prompting an expectation of many more ligands that mediate T-cell responses are waiting to be found. Using an unbiased mass spectrometry approach, here we identify pyridoxal (vitamin B6) as a naturally presented endogenous MR1 ligand, capable of activating the 7.G5 T-cell receptor, shown previously to recognise and kill cancer cells with high specificity. Subsequent metabolomic quantitation of pyridoxal and related vitamers in a panel of cancerous cells revealed vastly higher endogenous vitamin B6 levels compared to non-cancerous cell types, providing a potential mechanism for pyridoxal mediated cancer recognition by MR1-reactive T-cells.