Autonomous vehicles (AVs) offer numerous advantages, including improved mobility, enhanced comfort, and a reduction in traffic accidents. However, the acceptance and adoption of AVs by the public depend on their attitudes towards this emerging technology. The relationship between public knowledge of AVs and their attitudes has been a topic of debate, with conflicting findings. This study aims to examine the correlation between public knowledge, and attitudes towards AVs in the USA. A questionnaire survey was conducted between June and November 2022, collecting 5778 complete responses from diverse regions across the USA. The collected data were analysed to assess the public’s attitudes and knowledge levels in different regions. The findings revealed a negative shift in public attitudes towards AVs as knowledge levels increased. Specifically, a 1% increase in knowledge corresponded to a 0.65% decrease in interest, a 0.68% decline in trust, a $2466 USD reduction in willingness to pay for AVs, and a 0.56% increase in concerns about traveling in AVs. Furthermore, additional analyses were performed to explore how the attitudes of various demographic groups evolve with varying levels of knowledge showing a consistent negative shift in the attitude with the increase in the level of knowledge for participants across the different demographic groups.