Purpose To evaluate the ultra-morphology of the dentin after irradiation with Er:YAG laser using shorter pulse durations in different protocols by transmission electron microscopy (TEM). The Er:YAG laser is an effective alternative method for hard dental tissue treatment with great acceptability among patients. However, undesirable effects may occur in dental substrate if the correct parameters are not chosen during irradiation protocols, jeopardizing bonding that could result in premature failures. But, before doing bonding tests, it is fundamental the knowledge of the laser action into dentin. It is well known that shorter pulses have less thermal damage due to less heat loss during irradiation; hence, it is primordial to evaluate the performance of shorter pulses in irradiation protocols. Methods Eighteen dentin disks with 2 mm thick were assigned into six experimental groups according to the irradiation protocol: cavity preparation (200 mJ and 20 Hz) and pretreatment (80 mJ and 2 Hz) varying the pulse duration (50, 300, or 600 μs), in focused distance and under refrigeration. Qualitative and semi-quantitative analyses were performed. Results The pretreatment with 50 μs was the protocol that maintained intact both architecture of collagens fibrils and an organized extracellular matrix. Nevertheless, the other groups showed a melted and carbonized dentin surface followed by fused zone of collagen fibrils with loss of interfibrillar spaces. Conclusions It was concluded that the thickness of the altered layer is directly proportional to the enhance of pulse duration for cavity preparation protocols tested. Moreover, pretreatment with 50 μs is an adequate protocol for dentin, which did not alter its ultra-morphology.