H/ACA guide RNAs direct site-specific pseudouridylation of substrate RNAs by forming ribonucleoprotein (RNP) complexes with pseudouridine synthase Cbf5 and three accessory proteins. Recently determined crystal structures of H/ACA protein complexes and a fully assembled H/ACA RNP complex have provided significant insights into the architecture, assembly and mechanism of action of RNA-guided pseudouridine synthase. The binding of guide RNA is directed by its conserved secondary structure and sequence motifs, which enables guide RNA with different sequences to be incorporated into the same protein complex. Accessory proteins and peripheral domains crucially coordinate the position of guide RNA, and possibly regulate the reaction process.