RNA aptamer is one of functional RNAs selected experimentally from randomized RNA pools, and has a high affinity and specificity against the target such as a protein and a small ligand. For therapeutic use of RNA aptamer in vivo, both no side effects and escaping from RNase-induced degradation may be required. Here we show a first evidence of producing a circular streptavidin aptamer in vitro generated by self-splicing permutated intron-exon (PIE) sequences derived from gene td of T4 bacteriophage. In spite of its complex higher-order structure, the aptamer sequence was circularized. The circularized aptamer retains its binding affinity toward streptavidin and its binding was inhibited in the presence of biotin. The availability of circularizing the RNA aptamer in vitro would be a candidate for a new methodology for RNA therapeutics.