Morphological and chemical comparative analysis of the human and bovine dentin-adhesive layer. Micro energy-dispersive X-ray fluorescence spectrometry (µ-EDXRF) and scanning electron microscopy (SEM) were used to evaluate dentin treated with an etch and rinse adhesive (ER) and a self-etch adhesive (SE). Ten human molars (H) and ten bovine anterior teeth (B) were prepared (exposure of dentin and divided in the middle) and allocated into two different adhesion strategy groups per substrate (n=40). µ-EDXRF data and SEM images were obtained before and after treatment. Untreated dentin of both substrates did not differ in terms of Ca (p<0.1503), P (p<0.2986) or Ca/P ratio (p<0.1400). H-SE and B-SE specimens showed reduced P content (p<0.0001; p<0.0002), while H-ER and B-ER specimens showed reduced Ca and P content (p<0.0001; p<0.0001) when compared with untreated specimens. The Ca/P ratio was significantly higher in H-ER and B-ER specimens than in H-SE and B-SE specimens (p<0.0001; p<0.0080). Untreated dentin showed a homogeneous elemental distribution. However, after adhesive treatments, the surface of the dentin showed an irregular demineralization pattern. The resin tags and adhesive layer were shorter in bovine specimens than in human specimens due to morphological differences.