Introduction: Hormone treatment induces feminization of the body in transwomen and masculinization in transmen. However, the effect of hormone treatment on facial characteristics is still unknown. Aim: We aimed to study whether hormone treatment induces facial feminization and masculinization and how this potential change affects satisfaction and self-esteem. Methods: In this single-center cohort study, we included 27 transwomen and 15 transmen who received standardized hormone treatment in the Center of Expertise on Gender Dysphoria, VU University Medical Center Amsterdam. Facial 3-dimensional images were obtained at baseline and at 3 and 12 months. At each image, 22 facial landmarks were placed. Furthermore, the FACE-Q Satisfaction with Facial Appearance Overall and the Rosenberg self-esteem scale were obtained at the same measurement points. Main Outcome Measures: The main outcome measures included the relative local shift of skin in millimeters in the 22 landmarks in the transverse (x-axis), coronal (y-axis), and sagittal (z-axis) anatomic axes, the color maps, and the outcomes of the questionnaires. Results: After 12 months, cheek tissue in transwomen increased, with 0.50 mm (95% CI 0.04–0.96) in the x-axis and 1.08 mm (95% CI 0.31–1.85) in the z-axis. Tissue in the jaws decreased with −0.60 mm (95% CI −1.28–0.08) in the x-axis and −0.18 mm (95% CI −0.03–0.33) in the y-axis. Cheek tissue in transmen decreased with −0.45 mm (95% CI −1.00–0.11) in the x-axis and −0.84 mm (95% CI −1.92–0.25) in the z-axis. These changes already started after 3 months. An increase in satisfaction with the facial appearance was found in both transwomen and transmen. There were no changes in reported self-esteem. Clinical implication: These results could lead to more realistic expectations of facial changes. Furthermore, our results suggest that the face continues to change for at least a year, which could suggest that performing facial feminization surgery after 1 year of hormone treatment might be too early. Strength & Limitations: This study is the first that provides insight into the facial changes in transgender individuals receiving hormone treatment, and it introduces an objective method to examine (small) facial changes. Our study is limited by the poor reliability of the landmarks, the difficulty of facial fixation, and the lack of gender-specific questions in the questionnaires. Conclusions: Hormone treatment in transwomen induces an increase in cheek tissue and a decrease in jaw tissue. In transmen a tendency of decrease in cheek tissue and an increase in jaw tissue was found. These changes are in the direction of the desired gender. Tebbens M, Nota NM, Liberton NPTJ, et al. Gender-Affirming Hormone Treatment Induces Facial Feminization in Transwomen and Masculinization in Transmen: Quantification by 3D Scanning and Patient-Reported Outcome Measures. J Sex Med 2019;16:746–754.