This study focuses on the improvement of pre-service teachers’ self-efficacy for teaching science by including science courses within the teacher training program. Knowing how efficacy beliefs change over time and what factors influence the development by pre-service primary teachers of positive science teaching efficacy beliefs may be useful for teacher training universities, so that they can adapt their curriculum to accommodate these factors. Participants included 292 pre-service primary teachers, a cross-sectional sample from two different universities in the Netherlands across the four different years of study in the training program. Based upon our results, we conclude that the science teaching self-efficacy of pre-service teachers, in particular, improved during years 1 and 2, and not during years 3 and 4. Higher levels of self-rated subject-matter knowledge and science teaching experience in primary schools both contributed to higher levels of personal self-efficacy for science teaching. Differences at the university level in courses taken during the first year between science content courses and science methods courses also influenced the pre-service teachers’ development of science teaching self-efficacy. After their first year, the pre-service teachers from the university with science content courses had significantly higher self-efficacy than pre-service teachers from the university that offered science methods courses. After the second year of teacher training, however, this difference in self-efficacy was no longer present.