Objectives <p>Root caries (RC) progression involves the dentin extracellular matrix (<i>d</i>ECM) degradation by proteolytic enzymes. As neutrophil-derived enzymes have been systemically associated with ECM degradation, we hypothesized that neutrophil activity would: (i) inhibit RC remineralization; and (ii) alter the caries-affected-like dentin.</p> Materials and methods <p>In vitro RC were created on human dentin specimens. Half each lesion was covered to keep the initial lesion (IL). The other half (final lesion; FL) was exposed to one of the following groups (<i>n</i> = 10): Control (buffer solution; C); Neutrophils (human neutrophils; N); Collagenase (positive control; Col), and then remineralized by pH-cycling and sodium fluoride. RC remineralization was calculated using rhodamine infiltration and cross-sectional microhardness (CSMH). Collagen of the fully demineralized dentin was stained with picrosirius red. The physical properties of the caries-affected-like dentin were investigated by CSMH and loss of mineral density (MD).</p> Results <p>Two-way ANOVA and Tukey showed only remineralization in the C group (<i>p</i> &lt; 0.001). N did not show any remineralization (<i>p</i> = 0.588) between IL and FL. The N and Col groups exhibited changes in collagen integrity and organization. The N and Col groups’ caries-affected-like dentin showed significantly lower CSMH (<i>p</i> &lt; 0.001) than the C group, and significant losses (IL-FL) of MD (<i>p</i> = 0.012 and <i>p</i> = 0.042, respectively). In contrast, no MD loss was observed in the C group (<i>p</i> = 0.411).</p> Conclusions <p>Neutrophil-derived enzymes compromise RC remineralization and detrimentally affect the physical properties of the caries-affected-like dentin.</p> Clinical significance <p>Identifying proteolytic sources involved with <i>d</i>ECM degradation during RC progression will pave new research directions.</p>