We assessed the anti-SARS-CoV-2 spike antibody response to the third dose of BNT162b2 mRNA COVID-19 vaccine in Japanese hemodialysis patients and determined factors associated with the anti-SARS-CoV-2 spike antibody titer after the third dose of COVID-19 vaccine. Methods: Overall, 64 patients were enrolled in this single-center, prospective, longitudinal study. Anti-SARS-CoV-2 spike antibody titers were compared between hemodialysis patients and 18 healthcare workers. Multiple linear regression analysis was used to identify factors associated with the anti-SARS-CoV-2 spike antibody titer after the third vaccination. Results: There was no significant difference in anti-SARS-CoV-2 spike antibody titer 4 weeks after the third vaccination between hemodialysis patients and healthcare workers (18,500 [interquartile range, 11,000–34,500] vs. 11,500 [interquartile range, 7,918– 19,500], all values in AU/mL; p = 0.17). Uric acid (standard coefficient [β] = −0.203, p = 0.02), transferrin saturation (β = −0.269, p = 0.003), and log–anti-SARS-CoV-2 spike antibody titer 1 week before the third vaccination (β = 0.440, p < 0.001) correlated with the log–anti-SARS-CoV-2 spike antibody titer 4 weeks after the third vaccination. In contrast, only the log–anti-SARS-CoV-2 spike antibody titer 1 week before the third vaccination (β = 0.410, p < 0.001) correlated with the log– anti-SARS-CoV-2 spike antibody titer 12 weeks after the third vaccination. Conclusion: The anti-SARS-CoV-2 spike antibody titer after the third dose of COVID-19 vaccine was comparable between hemodialysis patients and healthcare workers. Uric acid concentration, transferrin saturation, and anti-SARS-CoV-2 spike antibody titer before the third dose were associated with the anti-SARS-CoV-2 spike antibody titer after the third dose in Japanese hemodialysis patients.