Objective To understand direct medical expenses of patients with healthcare-associated infection (HAI) due to carbapenem-resistant Gram-negative organism (CRO) under different medical insurance payment modes. Methods Case control study was adopted, patients with CRO HAI in a tertiary first-class hospital from January 2019 to December 2021 were selected as infection group, after the 1 ∶1 conditional matching, control group was screened, length of hospital stay and average hospitalization expense of two groups of patients were described by median, direct medical expense of CRO HAI under different payment modes was analyzed by rank sum test. Results There were 75 CRO HAI patients in infection group; 75 cases were successfully matched as control group. Compared with control group, length of hospital stay in patients in infection group extended by 24.4 days, the average hospitalization expense increased by 104 000 Yuan, the average daily hospitalization expense increased by 6 000 Yuan, the self-paid expense amount increase by 71 000 Yuan, and expense of antimicrobial agents increased by 5 000 Yuan, differences were all significant (all P < 0.05). The average hospitalization expense in patients in infection group with different payment modes were significantly different from that of control group (all P < 0.05). The average hospitalization expense due to infection of municipal employee group increased most than that of control group, which was 145 000 Yuan; compared with control group, the average hospitalization expense due to infection of all self-paid group increased by 122 000 Yuan, the self-paid expense due to infection of patients with municipal residents medical insurance and provincial medical insurance increased by 55 000 Yuan and 14 000 Yuan respectively, with a significant difference (both P < 0.05), the average daily hospitalization expense due to infection of provincial employees increased by 3 000 Yuan, differences were all significant (all P=0.001). Conclusion Under different medical insurance payment modes, the average expense of each hospitalization, average length of hospital stay, average daily hospitalization expense and self-paid amount of CRO-infected patients are higher than those of non-infected patients, which cause a heavy economic burden on municipal employees, rural cooperative medical care and all self-paid patients.