This paper describes the co-design and development process of an evidence-informed e-training program (Sit-Stand e-Guide) to support the safe and optimal use of sit-stand workstations from ergonomics and behavioural change perspectives. Using an instructional system design process, supported by a participatory design approach, data was collected through three workshops with workplace consumers (staff [n = 5] and managers [n = 5]), and subject matter experts (n = 5). Content and learning activities were developed based on behaviour change principles and optimal pedagogy. Key topics identified for the e-training were sedentary behaviour and health; workstation set-up; and strategies for behaviour change. Learning activities (scenarios and reflection) to enhance knowledge retention and skills implementation and an interactive one-page guide on completion were included in the e-training. The relevance and usefulness of the training prototype were reviewed through one-to-one think-aloud sessions with the workshop consumers (n = 5) and external health and safety professionals (n = 5) receiving positive feedback. The Sit-Stand e-Guide is readily available for workplace implementation and evaluation. This paper serves as a practical guide for future training development.