Automated vehicles (AVs) are promoted with numerous benefits such as enabling driver-passengers to perform non-driving-related tasks (NDRTs) while increasing road safety and reducing the environmental impact. However, public acceptance will play an essential role in the (non-)adoption of AVs. As yet, there has been scant research on the acceptance of AVs differentiated by gender across the lifespan. Therefore, we developed a questionnaire on the acceptance of automated driving (QAAD) and queried 351 female and 374 male participants aged 18–96 years in Germany. Our findings reveal substantial gender differences indicating that women assign lower ratings to the factors on positive aspects of AVs (PRO), NDRTs, and on Early Adoption, and higher values to the factors on worries about AVs (CON) and Sustainability than men, respectively. Additionally, we found that younger people reported higher scores for PRO, NDRTs, and Early Adoption than older people. However, we observed an age group effect not for all levels of automated driving and not for Sustainability. The identified evidence suggests that it is essential to address women and men separately across the lifespan if car manufacturers want AVs to be accepted in the future. Our findings may serve as a guideline for research on AVs in developing countries.