Importance: Increased free thyroxine (FT ₄ ) and decreased thyrotropin are associated with increased risk of atrial fibrillation (AF) in observational studies, but direct involvement is unclear. Objective: To evaluate the potential direct involvement of thyroid traits on AF. Design, Setting, and Participants: Study-level mendelian randomization (MR) included 11 studies, and summary-level MR included 55114 AF cases and 482295 referents, all of European ancestry. Exposures: Genomewide significant variants were used as instruments for standardized FT ₄ and thyrotropin levels within the reference range, standardized triiodothyronine (FT ₃ ):FT ₄ ratio, hypothyroidism, standardized thyroid peroxidase antibody levels, and hyperthyroidism. Mendelian randomization used genetic risk scores in study-level analysis or individual single-nucleotide polymorphisms in 2-sample MR for the summary-level data. Main Outcomes and Measures: Prevalent and incident AF. Results: The study-level analysis included 7679 individuals with AF and 49233 referents (mean age [standard error], 62 [3] years; 15859 men [29.7%]). In study-level random-effects meta-analysis, the pooled hazard ratio of FT ₄ levels (nanograms per deciliter) for incident AF was 1.55 (95% CI, 1.09-2.20; P =.02; I ² = 76%) and the pooled odds ratio (OR) for prevalent AF was 2.80 (95% CI, 1.41-5.54; P =.003; I ² = 64%) in multivariable-adjusted analyses. The FT ₄ genetic risk score was associated with an increase in FT ₄ by 0.082 SD (standard error, 0.007; P <.001) but not with incident AF (risk ratio, 0.84; 95% CI, 0.62-1.14; P =.27) or prevalent AF (OR, 1.32; 95% CI, 0.64-2.73; P =.46). Similarly, in summary-level inverse-variance weighted random-effects MR, gene-based FT ₄ within the reference range was not associated with AF (OR, 1.01; 95% CI, 0.89-1.14; P =.88). However, gene-based increased FT ₃ :FT ₄ ratio, increased thyrotropin within the reference range, and hypothyroidism were associated with AF with inverse-variance weighted random-effects OR of 1.33 (95% CI, 1.08-1.63; P =.006), 0.88 (95% CI, 0.84-0.92; P <.001), and 0.94 (95% CI, 0.90-0.99; P =.009), respectively, and robust to tests of horizontal pleiotropy. However, the subset of hypothyroidism single-nucleotide polymorphisms involved in autoimmunity and thyroid peroxidase antibodies levels were not associated with AF. Gene-based hyperthyroidism was associated with AF with MR-Egger OR of 1.31 (95% CI, 1.05-1.63; P =.02) with evidence of horizontal pleiotropy (P =.045). Conclusions and Relevance: Genetically increased FT ₃ :FT ₄ ratio and hyperthyroidism, but not FT ₄ within the reference range, were associated with increased AF, and increased thyrotropin within the reference range and hypothyroidism were associated with decreased AF, supporting a pathway involving the pituitary-thyroid-cardiac axis.