A person diagnosed with gender dysphoria who was assigned female at birth (AFAB) may request a vaginectomy as part of gender-affirming treatment. The aim of this study was to investigate the impact of vaginectomy on symptoms of pelvic floor dysfunction (PFD). This is a cohort study on patient-reported symptoms of PFD in patients who were AFAB, diagnosed with gender dysphoria, and undergoing vaginectomy in a single surgical center. Patients responded to a questionnaire preoperatively and 1 year postoperatively. The questionnaire consisted of 33 questions, including a modified short-form version of the Pelvic Floor Distress Inventory (PFDI-20). Twenty-three consecutive patients were included in the study and 20 patients (87%) completed the 1-year follow-up. The preoperative median PFDI-20 score was 24 (0-114) compared with 32 (0-168) at the 1-year follow-up (P = 0.07). Patients who had previously undergone neophallus construction with a metoidioplasty (n = 15) had no significant change between the preoperative and the 1-year postoperative PFDI-20 score [median 17.5 (0-114) and 27.5 (0-145) (P = 0.65), respectively]; whereas those with a groin flap phalloplasty (n = 5) had a significant increase in reported symptoms [median 37 (10-95) and 124 (45-168), respectively (P = 0.04)]. Overall, vaginectomy could be performed without any major impact on symptoms of PFD. However, this seemed to be true mainly for patients with previous metoidioplasty, whereas patients with previous groin flap phalloplasty reported worsening of symptoms.