Toll-like receptors (TLRs) have crucial roles in innate immunity, functioning as pattern-recognition receptors. TLR13 recognizes a conserved sequence from bacterial 23S rRNA and then triggers an immune response. Here we report the crystal structure of the mouse TLR13 ectodomain bound by a 13-nt single-stranded (ss) RNA derived from 23S rRNA. The ssRNA induces TLR13 dimerization but assumes a stem-loop-like structure that is completely different from that in the bacterial ribosome but nevertheless is crucial for TLR13 recognition. Most of the RNA nucleotides are splayed out to make base-specific contacts with the concave surface of TLR13, and RNA-specific interactions are important to allow TLR13 to distinguish RNA from DNA. Interestingly, a viral-derived 16-nt ssRNA predicted to form a similar stem-loop-like structure also induces TLR13 activation. Together, our results reveal the structural mechanism of TLR13's sequence- and conformation-specific recognition of ssRNA.