Abstract Background Antibodies binding to domain I of β2-glycoprotein I (aDI) and activated protein C (APC) resistance are associated with an increased risk of thrombosis in cross-sectional studies. The objective of this study was to assess their predictive value for future thromboembolic events in patients with antiphospholipid antibodies (aPL) or antiphospholipid syndrome. Methods This prospective multicenter cohort study included consecutive patients with aPL or systemic lupus erythematosus. We followed 137 patients (43.5 ± 15.4 year old; 107 women) for a mean duration of 43.1 ± 20.7 months. Results We detected aDI IgG antibodies by ELISA in 21 patients. An APC sensitivity ratio (APCsr) was determined using a thrombin generation–based test. The APCsr was higher in patients with anti–domain I antibodies demonstrating APC resistance (0.75 ± 0.13 vs 0.48 ± 0.20, P < 0.0001). In univariate analysis, the hazard ratio (HR) for thrombosis over time was higher in patients with aDI IgG (3.31 [95% CI, 1.15–9.52]; P = 0.03) and patients with higher APC resistance (APCsr >95th percentile; HR, 6.07 [95% CI, 1.69–21.87]; P = 0.006). A sensitivity analysis showed an increased risk of higher aDI IgG levels up to HR 5.61 (95% CI, 1.93–16.31; P = 0.01). In multivariate analysis, aDI IgG (HR, 3.90 [95% CI, 1.33–11.46]; P = 0.01) and APC resistance (HR, 4.98 [95% CI, 1.36–18.28]; P = 0.02) remained significant predictors of thrombosis over time. Conclusions Our study shows that novel tests for antibodies recognizing domain I of β2-glycoprotein I and functional tests identifying APC resistance are significant predictors of thrombosis over time and may be useful for risk stratification.