This study investigated psychological responses to AI-generated video ads, integrating the Uncanny Valley hypothesis with the Big Five personality traits. It focused on how perceived unnaturalness in audiovisual elements creates discomfort, influencing viewers’ advertising attitudes and behavioral intentions. For the study, 300 adults viewed an AI-produced public service announcement on environmental protection via an online survey. A series of regression analyses revealed that viewer discomfort was primarily triggered by auditory unnaturalness, while the direct impact of visual unnaturalness was relatively insignificant. Furthermore, personality factors such as neuroticism and openness were significant variables influencing the experience of discomfort. These traits also had an independent effect on how discomfort impacted advertising attitudes. Confirming established theories, ad attitude was a significant predictor of behavioral intention, a relationship also found to be multi-dimensionally influenced by respondent characteristics. This research offers theoretical insights for human-AI interaction and provides practical guidance for effective AI ad production.