Four of the five arginine tRNAs in E. coli contain a conserved 2‐thiocytidine (s2C) at position 32. The structural implications of this modification and how it impacts ribosomal binding are key to understanding its role in translation. The hypothesis that s2C modification creates specific structural changes in the anticodon stem and loop (ASL) of tRNAArg that result in functional binding differences is tested herein. Using electophoretic mobility, UV melt, circular dichroism, NMR and filter binding assays, we are able to develop structural and functional profiles of an array of unmodified and s2C‐containing arginine ASLs. It is shown that by combining the structural results with binding data creates a comprehensive view of how the complex dynamics of the translational machinery is influenced by the presence of s2C at position 32 of ASLArg. This study is funded by the National Science Foundation.