Recently, it was determined that representatives of the riboswitch candidates called ykkC and ykkC-III directly bind free guanidine. Guanidine-binding ykkC motif RNAs, now renamed guanidine-I riboswitches, were demonstrated to commonly regulate the expression of genes encoding guanidine carboxylases, as well as others encoding guanidine efflux proteins such as EmrE and SugE. Likewise, genes encoding similar efflux proteins are associated with ykkC-III motif RNAs, which have now been renamed guanidine-III riboswitches. Prior to the validation of guanidine as the ligand for these newly established riboswitch classes, another RNA motif was discovered by comparative genomic analysis and termed mini-ykkC because of its small size and gene associations similar to those of the original ykkC motif. It was hypothesized that these distinct RNA structures might respond to the same ligand. However, the small size and repetitive nature of mini-ykkC RNAs suggested that it might respond to ligand via the action of a protein factor. Herein, we demonstrate that, despite its extremely simple architecture, mini-ykkC motif RNAs constitute a distinct class of guanidine-sensing RNAs, called guanidine-II riboswitches. Surprisingly, each of the two stem-loop structures that comprise the mini-ykkC motif appears to directly bind free guanidine in a cooperative manner. These findings reveal that bacteria make extensive use of diverse guanidine-responsive riboswitches to overcome the toxic effects of this compound.