Objective: To evaluate the effectiveness of eradication therapy based on Helicobacter pylori (Hp) susceptibility and CYP2C19 genotype in children with refractory Hp infection. Methods: In this prospective observational cohort study, 156 children with Hp refractory to amoxicillin+clarithromycin+omeprazole triple regimen in Baoding Children′s Hospital from December 2017 to May 2018 were enrolled. Ninety-two of them underwent Hp culture and CYP2C19 detection. Seventy-five cases with positive Hp culture were defined as culture successful group and were treated according to Hp susceptibility and CYP2C19 genotype. Seventeen cases with negative Hp culture were defined as culture failed group and were treated only based on the results of CYP2C19 genotype. Sixty-four children who did not have Hp culture and CYP2C19 gene testing were defined as the empirical eradication therapy group and were treated with quadruple regimen (amoxicillin+metronidazole+omeprazole+bismuth). Bacterial resistance, CYP2C19 polymorphism and therapeutic effectiveness between the three groups were compared using chi-square test. Results: Among the 75 positive Hp culture results, 72 (96%) were resistant to clarithromycin, 3 (4%) were resistant to metronidazole, 5 (7%) were resistant to levofloxacin, 5 (7%) were resistant to rifampicin, 1 (1%) was resistant to tetracycline, and none was resistant to amoxicillin and furazolidone. The CYP2C19 polymorphism in 92 patients showed that 43 (47%) were extensive metabolizer (EM), 9 (10%) were poor metabolizer (PM), and 40 (43%) were intermediate metabolizer (IM). In terms of the effectiveness, eradication rate in the culture successful group,culture failed group and empirical eradication therapy group were 99% (74/75), 88% (15/17) and 72% (46/64), respectively (χ²=21.325, P<0.05). The eradication rate in the culture successful group was significantly higher than that in empirical eradication therapy group (χ²=21.005, P<0.05), while there was no difference between empirical eradication therapy group and culture failed group (χ²=1.154, P=0.283). Conclusion Eradication regimen based on bacterial susceptibility and CYP2C19 genotype should be considered in children with refractory Hp infection.