The mineral and organic phases of mineralized dentin contribute co-operatively to its strength and toughness. This study tested the null hypothesis that there is no difference in nano-dynamic mechanical behavior (complex modulus-E*; loss modulus-E''; storage modulus-E'; in GPa) of dentin hybrid layers (baseline: E*, 3.86 ± 0.24; E'', 0.23 ± 0.05; E', 3.85 ± 0.24) created by an etch-and-rinse adhesive in the presence or absence of biomimetic remineralization after in vitro aging. Using scanning probe microscopy and nano-dynamic mechanical analysis, we demonstrated that biomimetic remineralization restored the nano-dynamic mechanical behavior of heavily remineralized, resin-sparse regions of dentin hybrid layers (E*, 19.73 ± 3.85; E'', 8.75 ± 3.97; E', 16.02 ± 2.58) to those of the mineralized dentin base (E*, 19.20 ± 2.42; E'', 6.57 ± 1.96; E', 17.39 ± 2.0) [p > 0.05]. Conversely, those resin-sparse, water-rich regions degraded in the absence of biomimetic remineralization, with significant decline [p < 0.05] in their complex and storage moduli (E*, 0.83 ± 0.35; E'', 0.88 ± 0.24; E', 0.62 ± 0.32). Intrafibrillar apatite deposition preserves the integrity of resin-sparse regions of hybrid layers by restoring their nanomechanical properties to those exhibited by mineralized dentin.