Non-universal genetic codes are frequently found in animal mitochondrial decoding systems. In squid mitochondria, four codons deviate from the universal genetic code, namely AUA, UGA, and AGA/AGG (AGR) for Met, Trp, and Ser, respectively. To understand the molecular basis for establishing the non-universal genetic code, we isolated and analyzed five mitochondrial tRNAs from a squid, Loligo bleekeri. Primary structures of the isolated tRNAs, including their post-transcriptional modifications, were analyzed by mass spectrometry. tRNA(Met)(AUR) possessed an unmodified cytidine at the first position of the anticodon, suggesting that the AUA codon is deciphered by CAU anticodon via non-canonical A-C pairing. We identified 5-taurinomethyluridine (τm(5)U) at the first position of the anticodon in tRNA(Trp)(UGR). τm(5)U enables tRNA(Trp) to decipher UGR codons as Trp. In addition, 5-taurinomethyl-2-thiouridine (τm(5)s(2)U) was found in mitochondrial tRNAs for Leu(UUR) and Lys in L. bleekeri. This is the first discovery of τm(5)U and τm(5)s(2)U in molluscan mitochondrial tRNAs.