Objective To analyze the clinical infection characteristics, detection rates of antimicrobial resistance and virulence genes, as well as biofilm formation ability of T6SS-positive carbapenem-resistant Klebsiella pneumoniae (CRKP), provide reference for clinical prevention and control of CRKP infection. Methods Clinically isolated CRKP strains and patients' data from a tertiary first-class hospital in Anhui Province from January 2019 to December 2022 were collected. T6SS genes, virulence genes, antimicrobial resistance genes, and molecular typing were detected with polymerase chain reaction method. Biofilm formation ability was detection with 96-well plate crystal violet staining method. Results A total of 160 CRKP strains were included in study. The main sources of specimens were sputum (46.9%) and blood (26.3%). CRKP strains exhibited multidrug-resistant phenotype, mainly carrying bla_KPC (80.6%), followed by bla_NDM (17.5%). CRKP were divided into T6SS-positive group (n=129, 80.6%) and T6SS-negative group (n=31, 19.4%) based on whether they carried T6SS. The proportion of patients with chronic pulmonary and heart diseases in T6SS-positive group was higher than that in T6SS-negative group (P < 0.05), and the prognosis was worse than negative group (P < 0.05). In T6SS-positive group, the detection rates of iucA, mrkD, rmpA2, peg344, wabG, and fimH were all higher than those in T6SS-negative group (all P < 0.05). The main type in CRKP was ST11 (68.8%), with K64-ST11 accounting for 70.9% and K47-ST11 accounting for 25.5%. The proportions of ST11 CRKP and K64-ST11 CRKP in T6SS-positive group were both higher than T6SS-negative group (both P < 0.05). Biofilm formation ability of CRKP in T6SS-positive group was stronger than T6SS-negative group (P < 0.001). Except bla_OXA-48 gene, there was no statistical difference in carrying of other carbapenem resistance genes and antimicrobial resistance rates between the two groups. Conclusion CRKP in this area exhibits multidrug resistance, with a higher detection rate of T6SS. T6SS-positive CRKP has a higher detection rate of virulence genes and stronger biofilm formation ability.