<p>Collagen deposition and alignment in the tumor stroma stiffen the extracellular matrix and, via integrin signaling, stimulate proton extrusion by cancer cells. In aggressive tumors, migration occurs preferentially along aligned collagen fibers, where proton efflux at the leading edge acidifies and degrades the matrix to facilitate invasion. Whether directional proton diffusion along collagen fibers exacerbates invasion remains uncertain, as it is not known how far protons can travel along a fiber before being released. To characterize the release barrier, we labeled collagen lysine side chains with a pH-sensitive fluorescein derivative and introduced an interfacial proton flux by microinjecting HCl near the surface of self-assembled collagen fibers elevated above the chamber bottom. Proton wave propagation was monitored by wide-field fluorescence microscopy. Quantitative analysis of the fluorescence transients revealed that proton migration is best described by a one-dimensional diffusion model with a finite surface-to-bulk release rate. A single global parameter set consistently described all experiments. From the fitted release rate, we determined a Gibbs activation energy barrier Δ<i>G</i>^‡ of approximately 30 <i>k</i>_B<i>T</i>, comparable to values reported for membrane–water interfaces. These results demonstrate that protons can travel tens of micrometers along collagen fibers before equilibrating with the bulk solution, suggesting that membrane-anchored collagen fibers may provide an effective pathway for directing acidification along migration tracks in tumor stroma.</p>