Bovine lactoferrin (LF) is subjected to thermal processing during isolation for commercial use and while preparing milk products intended for infant nutrition. The present study is focused on the heat-induced structural changes of LF in buffer solution. Fluorescence spectroscopy, molecular modeling, and enzymatic hydrolysis studies were combined to extensively characterize LF thermal behavior. The temperature-induced changes induced on LF conformation were analyzed through intrinsic and ANS fluorescence parameters (intensity, maximum position, and parameter A value), the phase diagram method, and quenching experiments using acrylamide and iodide. A higher exposure of hydrophobic residues was highlighted through the molecular modeling approach, with a decrease in α-helix content from 23.5% to 21.2% when increasing the temperature from 25 °C to 80 °C. The experimental results demonstrate a more flexible conformation of the protein at higher temperature, thus facilitating the enzymatic hydrolysis by thermolysin.