To examine the value of the crossover sign (COS) in predicting treatment outcome in women with a Cesarean scar pregnancy (CSP) who were treated with ultrasound-guided vacuum aspiration. This was a retrospective cohort study of women with CSP who underwent ultrasound-guided vacuum aspiration. Based on the relationship between the gestational sac, Cesarean scar and anterior wall of the uterus, CSPs were classified by COS type. Analysis was conducted to investigate the association between COS type (COS-1, COS-2) and treatment outcome. The incidence of treatment failure, retained pregnancy tissue, secondary therapy and bleeding ≥ 200 mL were analyzed. In total, 181 eligible patients with CSP, including 90 (49.7%) women with COS-1 and 91 (50.3%) women with COS-2, were analyzed. COS-1 patients had a higher incidence of treatment failure compared with COS-2 patients (25.6% vs 8.8%; P = 0.003), as well as higher rates of retained pregnancy tissue (18.9% vs 6.6%; P = 0.013), secondary therapy (20.0% vs 6.6%; P = 0.002) and bleeding of ≥ 200 mL (13.3% vs 4.4%; P = 0.034). COS-1 and a large gestational sac (30.1-50.0 mm or >50.0 mm in diameter) were associated independently with increased risk of treatment failure (odds ratio, 4.57 (95% CI, 1.66-12.56); P = 0.003, 4.34 (95% CI, 1.35-13.94); P = 0.014 and 10.50 (95% CI, 2.54-43.46); P = 0.001, respectively). Ultrasound evaluation of the relationship between the gestational sac and the endometrial line (COS classification) in women with CSP may help to predict treatment outcome among those undergoing vacuum aspiration. Among COS-1 patients, especially those with a gestational sac diameter of >30.0 mm, vacuum aspiration may be discouraged. © 2023 International Society of Ultrasound in Obstetrics and Gynecology.