Objective To evaluate the relationship between the use of proton pump inhibitors (PPIs) and the occurrence of healthcare-associated infection (HAI)in hospitalized patients, providing a theoretical basis for the prevention and control of HAI. Methods HAI cases reported from 3 public hospitals of different classes in Gansu Province between January 2023 and December 2024 were selected as the case group. The stratified random sampling method was adopted, the case group was divided into four levels based on the Charlson comorbidity index (CCI). An equal number of cases without HAI during the same period were selected as control group through CCI stratification, and a retrospective case-control study was conducted. The risk factors for HAI as well as the dose-response relationship between duration of PPI use and HAI occurrence were explored. Results The use rate of PPIs in patients in the case group was higher than that in the control group (45.36% vs 29.57%), with a statistically significant difference (P<0.001, OR=1.98). Multivariate logistic regression results indicated that the adjusted OR value for use of PPIs was 1.78 (P<0.001), its infection risk was comparable to that of invasive procedures such as urinary catheterization. The median duration of PPI use in patients in the case group was 9 days, which was longer than the 8 days in the control group (P<0.001). An extension of duration of PPI use and increasing of infection risk presented a dose-response relationship (OR=4.59 when the duration of PPI use exceeded 14 days). Additionally, the proportion of oral combined with intravenous administration in patients in the case group was higher than that in the control group, and urinary catheterization and central venous catheterization also increased the risk of HAI (all P<0.05). Conclusion The use of PPIs in hospitalized patients can increase the risk of HAI, and this risk rises with prolonged medication duration. Factors such as administration method, length of hospital stay, as well as urinary catheterization and central venous catheterization also impact the occurrence of HAI.