The present study aims to examine the effect of the life skills training guide on the development of pre-service science teachers’ professional knowledge of entrepreneurial skills. The study included 82 pre-service science teachers studying in the third year of a public university in Turkey during the 2019-2020 academic year. The data of the study, which was conducted with a one-group pre-test-post-test experimental design, were collected using the entrepreneurship skills professional knowledge test. The data were analyzed using the professional knowledge assessment rubric and entrepreneurship skill indicators. The results of the study revealed that there was a significant difference between the pre-and post-test scores of the pre-service teachers in favor of the post-test scores. Following the experimental process, their professional knowledge about entrepreneurship increased. However, the number of pre-service teachers who were able to improve their professional knowledge scores to a high level was limited. Furthermore, following the experimental process, there was an increase in the number of pre-service teachers who were able to develop appropriate activities for students to gain entrepreneurial skills and to prepare tools for measuring and evaluating students’ entrepreneurial skills in a way to include stressed indicators. Consequently, the present study provides science teacher educators with educational activities that they can model within the scope of teaching professional knowledge of entrepreneurship skills to pre-service science teachers.