Introduction: COVID-19 vaccination in the United States (U.S.) has stalled, with the lowest rates observed in the southern parts of the country. Vaccine hesitancy is a primary contributor to this trend, and health literacy (HL) has been identified as a potential determinant for hesitancy. This study aims to assess the association between HL and COVID-19 vaccine hesitancy in a population residing in 14 southern states of the U.S. Methods: We utilized a web-based survey conducted in U.S. southern states between February and June 2021 that yielded 417 total responses. The outcome was “vaccine hesitancy”. The main independent variable was HL, assessed by validated measures whose responses were aggregated to an HL index score and subsequently categorized as “low or moderate HL”, and “high HL”. Descriptive statistics were performed, and multivariable logistic regression analysis was conducted, controlling for sociodemographic and other variables. Results: Of the total respondents (n=247 after data cleaning), the overall rate of vaccine hesitancy was 22.3%. The rate of vaccine hesitancy was 10.9% and 89.1% for low/moderate and high levels of HL, respectively, but the association was not significant. However, personal perception of COVID-19 threat was significantly associated with lower odds of vaccine hesitancy versus those without perception of threat [adjusted odds ratio, AOR: 0.15; 95% CI: 0.03-0.73; p = 0.0186]. Similarly, obtaining information from public health sources was significantly associated with a lower likelihood of vaccine hesitancy [AOR:0.17; 95% CI: 0.05-0.61; p=0.0064]. Conclusion: HL was not a significant predictor of vaccine hesitancy in a population residing in southern states, suggesting that low rates of vaccination in the region may not be due to knowledge about COVID-19. Personal perception of COVID-19 threats and obtaining information from public health sources were associated with decreased vaccine hesitancy. This implies that we should strengthen public health communication infrastructure in the country. We also need to unpack the contextual factors that contribute to the disproportionately high rates of vaccine hesitancy in the region.