This study investigated the effect of conventional and acidified casein phosphopeptide-amorphous calcium phosphate–containing crèmes (CPP-ACP and ACPP-ACP) on dentin permeability (DP) before and after acid challenge, using a fluid-flow measuring device and scanning electron microscopic (SEM) examination. Fifty dentin discs were prepared from intact human third molars. Each tooth was sectioned 3 mm above and below the cementoenamel junction. The smear layer was removed to expose the dentin and dentinal tubules. For fluid-flow measurement, 20 specimens were used and divided into two groups: (a) CPP-ACP (pH=7) and (b) ACPP-ACP (pH=2). The dentin surfaces were treated with the crème for five minutes daily for two weeks, and then an acid challenge was performed using 6% citric acid. Under simulated pulpal pressure, DP was measured at baseline, five minutes, two weeks, and after acid challenge. The remaining specimens were prepared and examined using SEM to investigate dentinal tubule occlusion. DP was not significantly different between the two CPP-ACP–containing crèmes at any test period (p≥0.05). DP after two weeks or acid challenge was significantly lower than that at five minutes (p<0.05). The results were consistent with the SEM micrographs that presented patent dentinal tubules after a five-minute application and partial blockage of dentinal tubules after the two-week application. The dentinal tubules remained partially occluded after acid challenge. Using conventional and acid-modified CPP-ACP–containing crèmes for two weeks decreased DP and partially occluded the dentinal tubules, which resisted acid challenge.