Objective To explore methods for improving the colonization efficiency of Helicobacter pylori (Hp) in the mouse stomach and to determine whether the proton pump inhibitor (PPI) pantoprazole can act as a colonization adjuvant to enhance Hp colonization. This study aims to provide an effective tool for establishing an Hp infection mouse model. Methods The Hp Sydney strain (SS1) was introduced, and solid plate and liquid culture systems were established and identified. The effects of different doses of pantoprazole on gastric acid secretion in mice were compared. The impact of Hp inoculation alone or combined with pantoprazole pretreatment on Hp colonization efficiency was analyzed using rapid urease tests, bacterial plate cultures, and TaqMan qPCR. Results Proton pump inhibitor pretreatment inhibited gastric acid secretion and, to some extent, promoted Hp colonization in the mouse stomach. Conclusion Proton pump inhibitors can serve as colonization adjuvants to enhance the efficiency of constructing Hp infection mouse models.