Uptake of a booster dose of COVID-19 vaccine is effective in preventing infection and severe consequences caused by COVID-19. The present study examined the effects of negative attitudes towards vaccination in general and trust in government on uptake of a COVID-19 booster dose, as well as the moderating role of psychological reactance to pro-vaccination messages in Hong Kong. An observational prospective cohort study using online survey was conducted among 264 adults. Findings showed that, after adjustment for significant background characteristics, negative attitudes towards vaccination in general negatively predicted uptake of a booster dose, and trust in government positively predicted uptake of a booster dose. In addition, the association between negative attitudes towards vaccination in general and uptake of a booster dose was weaker among those who reported a higher level of psychological reactance. The present study highlighted the importance of improving attitudes towards vaccination in general especially among those who are not experiencing psychological reactance, and building trust in government. This study also suggested that interventions aimed at improving attitudes towards vaccination in general should seek to avoid psychological reactance, and special attention should be given to people who are experiencing psychological reactance to pro-vaccination messages.