Abstract Exploring how science teacher education programs can prepare science teachers to support gender and sexually diverse students remains an important area for research. A 5‐week intervention was designed for pre‐service science teachers' (PSSTs), addressing gender and sexual diversity (GSD). The effects of the intervention on PSSTs' attitudes and beliefs about GSD‐inclusive science teaching (GSDST) were explored using a multiple case study research design. In addition, the design elements of the intervention that were perceived as most significant were identified. Our results showed that the PSSTs were mostly supportive of measures indicative of GSDST prior to the intervention, and there was an overall trend in favor of GSDST with small effect sizes after the intervention, which did not reach statistical significance. Using thematic analysis, three themes were identified to characterize how their attitudes and beliefs changed throughout the intervention: GSDST is perceived as important for student safety; an “add LGBT and stir” approach to GSDST; and uncertainty of GSD language. Five design features of the intervention that were perceived as most impactful were group dialog; coherence to Ambitious Science Teaching; GSD terminology; knowledge of intersex, hormones, and lesbian, gay, bisexual, transgender, and queer scientists; and relevant case studies. The findings contribute to understanding how science teacher education programs can impact PSSTs' attitudes, beliefs, and intended enactment of GSDST consistent, with recent calls for GSD equity in science education.