Background Humoral response against SARS-CoV-2 after two doses of BNT162b2 (Pfizer-BioNTech) has been proven less intense in maintenance dialysis patients as compared to healthy subjects, leading the French authorities to recommend a third injection in this population. Here, we investigated the response to the third injection in two cohorts of hemodialysis patients. Methods Data from two prospective observational cohorts were collected. In the first -“systematic”- cohort, patients from 2 hemodialysis centers (n = 66) received a 3rd injection of BNT162b2, regardless of the response after two injections. In the second -“conditional”- cohort, the injection was only prescribed to patients (n = 34) with no or low response to the previous two doses. In both cohorts, the 3rd dose was injected 1 to 2 months after the 2nd dose. Serology was performed after the 2nd and 3rd dose to assess anti-Spike IgG antibody titer. Results In the “systematic” cohort, anti-Spike IgG were found in 83.3% and 92.4% of patients after the 2nd and the 3rd dose of BNT162b2, respectively. In this cohort, 6/11 (54.5%) and 20/21 (95.2%) patients switched from non-responder to low-responder and from low-responder to high-responder, respectively. In low and high-responders to 2 doses, 50/55 (90.9%) at least doubled their anti-Spike IgG titer. Similar trends were observed in the “conditional” cohort. Conclusions In maintenance hemodialysis patients, humoral response against SARS-Cov-2 was boosted after a third dose of BNT162b2, allowing seroconversion in more than half of non-responders. These data may support an intensified vaccination protocol with a 3rd dose of BNT162b2 in dialysis patients.