OBJECTIVES: This study aimed to characterize the co-occurrence of the tigecycline resistance determinants tet(X4) and tmexCD2-toprJ2 in a Raoultella ornithinolytica isolate collected from a pig rectal swab at the slaughterhouse. METHODS: The R. ornithinolytica isolate WS60 was subjected to antimicrobial susceptibility testing. Whole-genome sequencing (WGS) was performed to analyze the genetic features of the plasmids carrying tet(X4) and tmexCD2-toprJ2. Additionally, a conjugation assay was conducted to evaluate the transferability of these plasmids, followed by a 15-day stability test to assess the persistence of the two resistance determinants. RESULTS: R. ornithinolytica WS60 exhibited high-level tigecycline resistance, with a minimum inhibitory concentration (MIC) of 32 μg/mL, and was also resistant to ampicillin, ampicillin-sulbactam, chloramphenicol, tetracycline, sulfamethoxazole-trimethoprim, florfenicol, and streptomycin. WGS analysis revealed that WS60 harbored three plasmids, including a 384,249-bp tmexCD2-toprJ2-carrying IncQ plasmid (pWS60-1) and a 78,159-bp tet(X4)-carrying IncFII plasmid (pWS60-2). Interestingly, pWS60-2 was identical to several plasmids found in Klebsiella spp. isolated from animals, animal-derived food, and humans. Moreover, pWS60-2 was successfully transferred to Klebsiella spp. via conjugation, whereas pWS60-1 failed to transfer. Notably, no significant fitness cost was observed in the transconjugants carrying pWS60-2. Additionally, a 15-day stability assay demonstrated that both resistance determinants were stably maintained in the bacterial population without significant loss, underscoring their persistence over time. CONCLUSIONS: This is the first report of the co-occurrence of tet(X4) and tmexCD2-toprJ2 in R. ornithinolytica. Enhanced surveillance in slaughterhouses, along with targeted interventions, should be implemented to mitigate the potential spread of mobile tigecycline resistance throughout the food production chain.