Introduction. Hepatitis B virus (HBV) vaccination has decreased the overall incidence of HBV infection; however, approximately 5 to 10% of people are non-responders to the vaccination. This study investigated the factors associated with non-response to HBV vaccination, with an emphasis on vitamin D deficiency (VDD). Methods. This retrospective observational study focused on adult workers in a single heavy industry. Individuals with negative initial hepatitis B surface antibody (anti-HBs) levels prior to vaccination and who then received a two- or three-dose series of HBV vaccinations were enrolled. The study endpoint was failure to achieve a seroprotective antibody response, defined as an anti-HBs titer less than 10 mIU/mL. Propensity score matching (PSM) and binary logistic regression models were used to adjust the outcomes for other clinical characteristics. Results. Among 760 workers, 566 (74.5%) exhibited VDD. The non-response rates to HBV vaccination were 13.4% (76/566) and 5.7% (11/194) among workers with and without VDD, respectively (p = 0.005). Even after adjustment using PSM, VDD was still associated with a higher rate of response failure (adjusted odds ratio 2.74; 95% confidence interval 1.40–5.38, p = 0.003). The binary logistic regression model showed that VDD, older age, omission of the third vaccine dose, lower initial anti-HBs titer, and current smoking were associated with response failure. Conclusions. Our study suggests that VDD may impair the serologic response following HBV vaccination. Further research is needed to evaluate the effectiveness of vitamin D supplementation in increasing the response to HBV vaccination.