The advancement of technology in Industrial Revolution 4.0 requires modern learning sources such as electronic book (e-Book). One subject material that had been developed in e-Book was metacognitive learning strategies. This material is significant at enhancing student’s self-directed learning that led into autonomous learning. The developed e-Book had numbers of features and was validated and avowed to be practical in learning process. This study was objected to describe student’s self-regulation skills and learning outcomes after using e-Book of metacognitive learning strategies. This experimental study used one-shot case study using three experimental classes with no control group. 82 students enrolled at Department of Biology Education, Universitas Negeri Surabaya, were involved as the research samples. Data were collected using questionnaire named Self-Regulated Learning Interview Scale/SRLIS administered through surveyplanet and test. The obtained data were then analyzed using one-way Anova by the assistance of SPSS and descriptive statistics. The results showed that students’ self-regulation skills and learning outcomes in the three classes were not significantly different and considered in a high category. Therefore, e-Book of metacognitive learning strategies can be used to train student’s self-regulation skills during the learning process.