The thiamine pyrophosphate (TPP)-sensing riboswitch is the only riboswitch found in eukaryotes. In plants, TPP regulates its own production by binding the 3' untranslated region of the mRNA encoding ThiC, a critical enzyme in thiamine biosynthesis, which promotes the formation of an unstable splicing variant. In order to better understand the molecular basis of TPP-analogue binding to the eukaryotic TPP-responsive rioswitch, we have determined the crystal structures of the Arabidopsis thaliana TPP-riboswitch in complex with oxythiamine pyrophosphate (OTPP) and with the antimicrobial compound pyrimidine ring OTPP is stabilized in its enol form in order to retain key interactions with guanosine 28 of the riboswitch previously observed in the TPP complex. The structure of PTPP in complex with the riboswitch shows that the moiety of guanosine 60 undergoes a conformational change to cradle the pyridine ring of the PTPP. Structural information from these complexes has implications for the design of novel antimicrobials targeting TPP-sensing riboswitches.