There has been an expansion of repertoire of unnatural amino acids that can be incorporated into recombinant proteins in vivo, but all of them are limited to the elongation process of protein synthesis. However, even though the N-terminal position of proteins can be utilized for diverse purposes, in particular chemical modifications, no method has been reported for positioning unnatural amino acids at the translation initiation site. In this study, I have designed a protein initiation system active for unnatural amino acids by engineering an orthogonal tyrosyl-tRNA from Methanococcus jannaschii, which had been developed with aminoacyl-tRNA synthetases for incorporating unnatural amino acids for the translation elongation step. To make the system orthogonal to the natural one, one of stop codons, amber codon, was chosen for the new translation initiation system. A couple of tRNA variants were generated by modifying the acceptor stem, the anticodon loop, and the T loop, aiming making interactions with formyltransferase and initiation factor 2 without hurting the aminoacylation of unnatural amino acids. One engineered tRNA supported the initiation of protein synthesis with O-propagyl tyrosine (OpgY) with overexpression of the formyltransferase. The incorporation of OpgY was confirmed by mass spectrometry and N-terminal sequencing analyses. However, one of natural amino acids, gluamine, was also incorporated into the amber codon, even though its efficiency was lower than that of OpgY. To minimize the misincorporation, attempts have been made to engineer the tRNA further.