Purpose: The aim of this study was to evaluate the effect of Er:YAG laser ablation on the microstructure of the dentin/adhesive interface after acid-base challenge in vitro. Materials and Methods: Thirty-two extracted third molars were used. One pair of disk-shaped specimens was obtained from the midcoronal dentin of each tooth. One surface in each pair was subjected to Er:YAG laser irradiation, while the other was ground using 600-grit SiC abrasive paper. The specimens were assigned to 4 groups with 8 pairs in each group. A self-etching primer (Clearfil SE-Bond), a bonding adhesive (Clearfil SE-Bond), and a photocuring composite (Clearfil APX) were applied according to manufacturer's instructions to create dentin-resin "sandwiches" of each pair, except in group IV, where no primer was applied. The specimens were stored in demineralizing solution for 20 min in groups I and IV, 60 min in group II, and 120 min in group III. After immersion in 5% NaOCl, all the specimens were polished and argon-ion etched. Finally, the interfaces between the dentin and bonding agent were observed under a scanning electron microscope. Results: All specimens in groups I to III showed an acid-base resistant zone, except those of group IV, which debonded completely. A statistically significant difference was found in the thickness of the acid-base resistant zone between group I vs groups II and III for both abraded and Er:YAG-irradiated surfaces. There was no statistically significant difference in the thickness of the acid-base resistant zone between groups II and III for both conventionally prepared and Er:YAG-irradiated surfaces. Conclusion: The existence of an acid-base resistant zone following the application of the SE Bond system on both abraded and Er:YAG-lased human dentin, regardless of the acid-base challenge duration, was confirmed. Keywords: Er:YAG laser, acid-base resistant zone, self-etching primer, dentin, acid-base challenge