Histology is a science fundamental for life and health undergraduate courses. During its teaching and learning process, digital books (e-books) stand out by offering students more contact time with theoretical–practical content; however, the reliability of these must be examined. Therefore, this study aimed to validate an e-book, Histologia interativa: roteiros de estudos (interactive histology: study guide) for teaching and learning histology at the university level. A methodological validation study was performed, in which the data were collected by filling out an online form regarding the qualities from experts and about the review of the e-book's content. Seven experts participated in the study, the majority of them working as professors at universities in the morphofunction field. The content validity indices for item (I-CVI) and scale (S-CVI) were calculated. Values≥0.78 for I-CVI and≥0.90 for S-CVI/AVE were considered to have excellent content validity. Only two (6.06%) of 33 items did not achieve acceptable I-CVI values. Overall, S-CVI/AVE obtained a value of 0.955. In conclusion, the e-book evaluation has sufficient structural- and criterion-related validity. Furthermore, despite the experts’ suggestions, they favored the use of the e-book in the teaching and learning of histology at the university level.