In this article, we report the results of gas-phase IR spectroscopy of neutral glycylglycine (Gly-Gly) in the 700-1850 cm-1 frequency range. A combination of laser desorption, jet-cooling, and IR multiple-photon dissociation vacuum-ultraviolet (IRMPD-VUV) action spectroscopy is employed, together with extensive quantum chemical calculations that assist in the analysis of the experimental data. As a result, we determined that the most favorable conformer in the low-temperature environment of the supersonic jet is the nearly planar structure with two C5 hydrogen-bonding interactions. Calculations clearly show that this conformer is favored because of its flexibility (considerable entropy stabilization) as well as efficient conformer relaxation processes in the jet. To gain more understanding into the relative stability of the lowest-energy Gly-Gly conformers, the relative strength of hydrogen bonding and steric interactions is analyzed using the noncovalent interactions (NCI) approach.