Background: Initiating hemodialysis via an arteriovenous (AV) access is considered best practice for most patients. Despite the well-recognized advantages of AV access, 80% of U.S. patients start hemodialysis with a catheter. Limited patient knowledge about vascular access, among other factors, may play a role in this high rate. We used iterative stakeholder input to develop novel, mixed-media vascular access education materials and evaluated their preliminary acceptability. Methods: We conducted preliminary focus groups and interviews with key stakeholders to assess patient vascular access understanding and elicit perspectives on existing education materials. We then used stakeholder input to inform initial development and iterative updates to the content and design of an animated video and complementary brochure. Video development (scripting, storyboarding, animation) was guided by an evidence-based framework and two health behavior change models. We assessed acceptability of the completed materials with patients and medical providers/personnel via interviews. Results: Overall, 105 stakeholders participated in education materials development and review (80 patients/care partners, 25 medical providers/personnel). Preliminary qualitative work included 52 patients/care partners and 16 providers/personnel; video development included 28 patients/care partners and 9 providers/personnel. The video script, storyboards, and animation underwent 14, 4, and 9 stakeholder-guided iterations, respectively. Responsive changes included aesthetic modifications, technical updates, and content additions (e.g., HD circuit, access self-monitoring, enhanced patient testimonials). The final 18-minute video and complementary brochure define vascular access types, describe care processes, outline potential complications, and address common patient concerns. Interviews with 28 patients/care partners and 9 providers/personnel from diverse geographic regions revealed preliminary acceptability of and enthusiasm for the materials by patients and providers. Conclusions: In collaboration with key stakeholders, we developed mixed-media vascular access education materials that were well-received by patients and providers. Preliminary findings suggest that the materials are promising to improve vascular access understanding among patients.