Purpose: The purpose of this study was to explore the self-efficacy of elementary pre-service teachers regarding online science instruction. Methods: A survey was conducted with 89 elementary pre-service teachers enrolled at Education University A, located in a non-metropolitan area. Among them, 3 participants agreed to additional interviews. Descriptive statistical analysis was conducted for each item, t-test were performed based on gender and major, and qualitative text (keywords) analysis was conducted for open-ended items. Interviews were conducted to elicit responses for open-ended items and were interpreted inductively through keyword analysis. The survey items were measured on a Likert scale for 8 subcategories, and one open-ended item allowed participants to freely describe their thoughts. Results: The results interpreted that elementary pre-service teachers have relatively low self-efficacy regarding online science instruction. There were no significant differences based on gender, but there were statistically significant differences in some items based on major. Ultimately, this suggests that different approaches may be needed for online science instruction based on pre-service teachers' backgrounds. This study can be interpreted as an indicator of pre-service teachers' perceptions and readiness for online science instruction before entering the school field. Conclusion: It emphasizes the importance of specific instructional methods and strategies for online science instruction and proposes the need for practical education.