We investigated the gene regulatory mechanism of a previously engineered riboswitch +thiMN(15)#19 that turns on gene expression in response to thiamine pyrophosphate (TPP). In vitro enzymatic probing was performed to identify the secondary structures of the OFF conformations predicted by Mfold. Interestingly, enzymatic probing data of the riboswitch and its variants indicated that the riboswitch in its OFF state adopts two distinct structures. Moreover, further in vivo experiments suggested that both OFF structures contribute to the riboswitch function. A deeper understanding of how riboswitches function at the molecular level should enhance our ability to design synthetic riboswitches with new or improved characteristics.