The structural significance of the hypermodified nucleosides lysidine (k²C) and N⁶-(N-threonylcarbonyl) adenosine (t⁶A) present in the anticodon loop of E. coli tRNA^Ile has not been studied in detail theoretically at the atomic level. Hence, in the present paper, we investigated the conformational preferences of modified nucleosides k²C and t⁶A in the anticodon loop of tRNA^Ile using various quantum chemical methods. Multiple molecular dynamics simulation studies of the anticodon stem loop (ASL) of tRNA^Ile have also been made to see the solvation effect. The lysine moiety of lysidine orients back and forms a hydrogen bond with the 2′-hydroxyl group of the 34th ribose sugar whereas the t⁶A₍₃₇₎ side chain prefers a ‘distal’ conformation. This kind of conformation would be useful for recognition of the codons AUA instead of AUG. The t⁶A side chain prohibits canonical base pairing and maintains an anticodon open loop structure. The preferred conformations of k²C₍₃₄₎ and t⁶A₍₃₇₎ are stabilized by various intra as well as inter residual interactions within the anticodon loop which could be useful for maintaining a proper anticodon loop structure for smooth and in phase codon–anticodon interactions during the biosynthesis of proteins.