Amikacin is a 2-deoxystreptamine aminoglycoside antibiotic possessing a unique l-HABA (l-(-)-γ-amino-α-hydroxybutyric acid) group and applied in the treatment of hospital-acquired infections. Amikacin influences bacterial translation by binding to the decoding region of the small ribosomal subunit that overlaps with the binding site of aminoacylated-tRNA (A-site). Here, we have characterized thermodynamics of interactions of amikacin with a 27-mer RNA oligonucleotide mimicking the aminoglycoside binding site in the bacterial ribosome. We applied isothermal titration and differential scanning calorimetries, circular dichroism and thermal denaturation experiments, as well as computer simulations. Thermal denaturation studies have shown that amikacin affects only slightly the melting temperatures of the A-site mimicking RNA model suggesting a moderate stabilization of RNA by amikacin. Isothermal titration calorimetry gives the equilibrium dissociation constants for the binding reaction between amikacin and the A-site oligonucleotide in the micromolar range with a favorable enthalpic contribution. However, for amikacin we observe a positive entropic contribution to binding, contrary to other aminoglycosides, paromomycin and ribostamycin. Circular dichroism spectra suggest that the observed increase in entropy is not caused by structural changes of RNA because amikacin binding does not destabilize the helicity of the RNA model. To investigate the origins of this positive entropy change we performed all-atom molecular dynamics simulations in explicit solvent for the 27-mer RNA oligonucleotide mimicking one A-site and the crystal structure of an RNA duplex containing two A-sites. We observed that the diversity of the conformational states of the l-HABA group sampled in the simulations of the complex was larger than for the free amikacin in explicit water. Therefore, the larger flexibility of the l-HABA group in the bound form may contribute to an increase of entropy upon binding.