The demonstration of efficient single-mode fiber (SMF) coupling is a key requirement for the development of a compact, ultra-precise radial velocity (RV) spectrograph. iLocater is a next generation instrument for the Large Binocular Telescope (LBT) that uses adaptive optics (AO) to inject starlight into a SMF. In preparation for commissioning iLocater, a prototype SMF injection system was installed and tested at the LBT in the Y-band (0.970-1.065 μm). This system was designed to verify the capability of the LBT AO system as well as characterize on-sky SMF coupling efficiencies. SMF coupling was measured on stars with variable airmasses, apparent magnitudes, and seeing conditions for six half-nights using the Large Binocular Telescope Interferometer. We present the overall optical and mechanical performance of the SMF injection system, including details of the installation and alignment procedure. A particular emphasis is placed on analyzing the instrument's performance as a function of telescope elevation to inform the final design of the fiber injection system for iLocater.