The purpose of this study was to evaluate the acid resistance of erbium:yttrium-aluminum-garnet (Er:YAG) lased sub-surface dentin using a scanning electron microscope (SEM). Dentin disks were exposed to a single pulse of Er:YAG laser irradiation at 80 mJ/pulse under water spray, with the contact sapphire tip vertical to the dentin surfaces (n = 15). Five specimens from the laser-ablated dentin surfaces were observed. Ten specimens were crosscut, exposing the center of the lased point. For five of the specimens, the sub-surface of the lased dentin, with or without 10% phosphoric acid treatment, was evaluated. We used the remaining five specimens to analyze the degree of acid treatment on crosscut surface by observing the re-crosscut surfaces at the lased point. The irradiated dentin surfaces were irregular, scaly or flaky. Three sub-layers were observed in the sub-surface: a superficial, less decalcified layer; an intermediate most decalcified layer; and a deep, normal shade layer. Er:YAG laser irradiation affected the acid resistance of sub-surface dentin.