Vaccine hesitancy is an important public health issue referring to concerns about the safety and efficacy of vaccination. Within a framework, this study aimed to assess the cultural adaptation, validity, and reliability of the Greek version of the adult Vaccine Hesitancy Scale (aVHS) as well as to identify the determinants of vaccine hesitancy among a large regional population in central Greece. A cross-sectional study was conducted enrolling 300 adults who had received primary healthcare services in the Health Centers and Local Health Units of the Magnesia Region from October to December 2022. The aVHS and the Life Orientation Test-Revised (LOT-R) were used to identify vaccine hesitancy and the dispositional level of optimism, respectively. For survey translation, the procedure of forward and backward translation was followed. Also, the aVHS was tested in a pilot study with a sample of 18 responders. Construct validity and internal consistency reliability were investigated via exploratory and confirmatory factor analysis and Cronbach’s alpha coefficients, respectively. Simple and multiple linear regression analysis were used to determine predictors for vaccine hesitancy. Factor analyses indicated that the aVHS comprises two constructs (“lack of confidence” and “risk perception”) explaining 68.9% of the total variance. The Cronbach’s alpha of the total scale was 0.884, indicating its high internal consistency. Participants who lived in rural areas, had a lower annual income, and reported a lower level of optimism showed a higher lack of confidence in vaccination. On the other hand, people aged above 45 years old who had graduated from high school or elementary school and were unemployed showed greater aversion to the risks of side effects. Finally, certain socio-demographic characteristics were associated with vaccine hesitancy. Our data suggest that the aVHS is a valid and reliable instrument for measuring vaccine-related attitudes and perceptions in Greek society, providing meaningful insight into designing vaccination-related preventive interventions in the community.