Helicobacter pylori infection is one of the main causes of gastric cancer. thioredoxin-1 (Trx1) and arginase (RocF) expressed by H. pylori were found to be closely related to its pathogenicity. However, whether Trx1 and RocF can be used in clinical screening of highly pathogenic H. pylori and the pathogenesis of trx1 high expressing H. pylori remain still unknown. We investigated the expression level of H. pylori trx1 and H. pylori rocF in human gastric antrum tissues using reverse transcription and quantitative real-time PCR (RT-qPCR) and clarified the clinical application value of trx1 and rocF for screening highly pathogenic H. pylori. The pathogenic mechanism of Trx1 were further explored by RNA-seq of GES-1 cells co-cultured with trx1 high or low expressing H. pylori. Differentially expressed genes and signaling pathways were validated by RT-qPCR, Enzyme-linked immunosorbent assay (ELISA), western blot, immunohistochemistry and immunofluorescence. We also assessed the adherence of trx1 high and low expressing H. pylori to GES-1 cells. We found that H. pylori trx1 and H. pylori rocF were more significantly expressed in the gastric cancer and peptic ulcer group than that in the gastritis group and the parallel diagnosis of H. pylori trx1 and H. pylori rocF had high sensitivity. The trx1 high expressing H. pylori had stronger adhesion ability to GES-1 cells and upregulated the interleukin (IL) 23A/nuclear factor κappaB (NF-κB)/IL17A, IL6, IL8 pathway. H. pylori trx1 and H. pylori rocF can be used in clinical screening of highly pathogenic H. pylori and predicting the outcome of H. pylori infection. The trx1 high expressing H. pylori has stronger adhesion capacity and promotes the development of gastric diseases by upregulating the activation of NF-κB signaling pathway.