Abstract Tigecycline (TGC) and eravacycline (ERV) are critical last-resort antibiotics used to treat complicated infections caused by extensively drug-resistant Gram-negative bacteria particularly carbapenem- and colistin-resistant Enterobacterales. The recent emergence of plasmid-mediated TGC resistance gene tet(X) in Enterobacterales of both animal and human origins represents a significant public health threat. In this study, we characterized tet(X4)-bearing Escherichia coli isolates recovered from cattle fecal samples in Iran. A total of 395 fecal samples obtained from calves were screened for tigecycline-resistant (TGC-R) E. coli by inoculating in to selective culture media containing tigecycline. The presence of tet(X) gene among the recovered TGC-R enteric bacteria was assessed using PCR. Genetic relatedness of the tet(X)-bearing strains was analyzed via ERIC-PCR. Three tet(X)-bearing strains were further characterized by whole genome sequencing (WGS) using Illumina platform. The transferability and stability of tet(X)-bearing elements were evaluated by conjugation assay and successive subculturing on antibiotic-free culture media respectively. A total of five tet(X)-positive E. coli isolates exhibiting high-level resistance to tigecycline (MIC = 64 mg/L) and eravacycline (MIC > 8 mg/L) were recovered and categorized in to two groups (n = 4, n = 1) based on ERIC-PCR and antimicrobial susceptibility patterns. WGS analysis identified tet(X4) variant in three isolates, which belonged to sequence types ST224 (n = 2) and ST10 (n = 1). ResFinder database analysis revealed coexistence of tet(X4) with multiple antibiotic resistance genes including aadA, aph, bla CTX−M−15 and /or bla TEM−1B, floR, cmlA, dfrA, sul and qnrS/mutations in gyrA and parC genes. The tet(X4)-positive E. coli isolates contained the IncX1 and p0111 (strains B52 and R37) or IncX1, IncQ1, IncI1-I(α) and IncFII/IncFIA/IncFIB (strain M55) replicon types according to PlasmidFinder analysis. The tet(X4) gene was successfully mobilized to tigecycline -susceptible recipient E. coli isolates through conjugation assay and demonstrated high stability persisting over 10 consecutive passages in antibiotic-free media in both transconjugants and their donors. This study reports, for the first time in Iran, the emergence of transferrable high-level tigecycline/eravacycline resistance gene tet(X4) in E. coli isolates. Given the public health implications, control measures should be implemented to regulate the use of tetracyclines and potentially phenicols in food animals to prevent emergence and further transmission of such superbugs along the animal- environment -human chain.