Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3′ untranslated regions of poly(A)-less histone mRNAs. The 3′ hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein–RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3′ end. The 3′ hairpin and presumably HBP/SLBP are also required for nucleo-cytoplasmic transport, translation, and stability of histone mRNAs. RNA 3′ processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson–Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3′ hairpin with HBP, and thus the regulation of histone mRNA metabolism.