Objective To understand antimicrobial resistance and distribution of virulence genes of New Delhi meta-llo-β-lactamase-1 (NDM-1)-producing Enterobacter cloacae (ECL). Methods ECL isolated from the First Affiliated Hospital of Kunming Medical University from January 2017 to November 2020 were collected, the trial group was 29 strains of NDM-1-producing carbapenem-resistant ECL (CRECL), control group was 32 strains of CRECL without producing NDM-1 and 35 strains of carbapenem-sensitive ECL(CSECL). Strains were identified and performed antimicrobial susceptibility testing with matrix assisted laser desorption ionization time-of-flight mass spectrometer(MALDI-TOF MS)and automated microbial identification system, NDM-1 resistance genes and 24 pairs of virulence genes were detected by polymerase chain reaction, change in distribution of virulence genes was compared with Chi-square test. Results Isolation rate of NDM-1 gene of CRECL strain isolated in this hospital was 47.5%, NDM-1-producing CRECL showed multiple resistance to common antimicrobial agents. Among 96 ECL strains, detection rates of virulence genes acrA, tolC, wcaA, wcaM, and wza were 80.2%, 90.6%, 87.5%, 75.0% and 92.7% respectively, detection rates of clpB, icmf, and VasD/Lip genes were also above 60%, virulence genes such as escV, nleB, pet, and hlyA were not found. Detection rates of clpB, icmf, VasD/Lip, and acrA genes in NDM-1-producing CRECL group were higher than those in CRECL group without producing NDM-1, detection rates of clpB, icmf, and VasD/Lip genes in CRECL group were higher than those in CSECL group, difference was statistically significant (P < 0.05). Conclusion Antimicrobial resistance of NDM-1-producing CRECL is severe, and the carrying rate of virulence genes also increased, antimicrobial resistance and virulence gene distribution of bacteria should be considered in clinical medication.