The aim of this study was to evaluate the surface roughness and wettability of dentin following ultrashort pulsed laser ablation with different levels of fluence and pulse overlap (PO). Twenty-five extracted human teeth crowns were cut longitudinally into slices of approximately 1.5-mm thick and randomly divided into nine groups of five. Samples in groups 1 to 8 were ablated with an ultrashort pulsed laser through a galvanometric scanning system. Samples in group 9 were prepared using a mechanical rotary instrument. The surface roughness of samples from each group was then measured using a three-dimensional profile measurement laser microscope, and wettability was evaluated by measuring the contact angle of a drop of water on the prepared dentin surface using an optical contact angle measuring device. The results showed that both laser fluence and PO had an effect on dentin surface roughness. Specifically, a higher PO decreased dentin surface roughness and reduced the effect of high-laser fluence on decreasing the surface roughness in some groups. Furthermore, all ablated dentin showed a contact angle of approximately 0 deg, meaning that laser ablation significantly improved wettability. Adjustment of ultrashort pulsed laser parameters can, therefore, significantly alter dentin surface roughness and wettability.