Processing of precursors of nuclear cis-splicing tRNA has been considered to start with the end maturation at the acceptor stem, followed by the intron splicing. The sequences around the intron-exon junctions in pre-tRNAs generally form a bulge-helix-bulge (BHB) motif, which is specifically recognized by the tRNA-splicing endonuclease. In a unicellular red alga Cyanidioscyzon merolae, we have found that the maturation of circularly permuted pre-tRNAs, in which the 3’-half of the tRNA coding sequence lies upstream of the 5’-half starts with the processing of the BHB motif at the junctions of termini to form a circular RNA intermediate. This suggested that the processing of BHB motifs precedes that of the acceptor stem in C. merolae. Here, we performed sequencing analyses for processing intermediates of various types of intronic tRNAs by reverse transcription polymerase chain reaction (RT-PCR) followed by the sequencing of the PCR products to clarify maturation pathway. Results showed that the removal of introns occurred before the maturation of acceptor stem. Such non-canonical processing pathway may have been prerequisite for the development of permuted tRNA genes in C. merolae because permuted pre-tRNAs can be processed via a continuous RNA string, but not fragments, during maturation.