Introduction: Enterococci are considered opportunistic pathogens. However, they can serve as a reservoir of antibacterial resistance (ABR) traits and transfer these to humans through the food chain. Methods: Antibiotic susceptibility testing and multilocus sequence typing were used to characterize the ABR and population structure of 488 enterococcal isolates recovered along the food chain from four provincial-level administrative divisions (PLADs) in China. Results: Enterococcus faecalis ( E. faecalis) was the dominant species cultured from pig farms and retail meat, while Enterococcus faecium ( E. faecium) and Enterococcus casseliflavus were dominant in patients with diarrhea and retail fruits and vegetables, respectively. Approximately 67% of all Enterococcus isolates were multidrug-resistant (MDR). E. faecium resistance to ampicillin and penicillin was significantly higher than that of E. faecalis. The E. faecalis isolates exhibited substantially heterologous sequence types (STs), whereas E. faecium isolates were clearly divided into clonal complex (CC) CC17 and CC94 clades. E. faecium isolates were mainly detected in hospitalized children and were identified as the hospital-associated CC17 clade with ampicillin and penicillin resistance. Notably, E. faecalis ST16 and ST65 and E. faecium ST60 and ST94 detected in patients with diarrhea were also detected in farm and food samples, indicating that these STs should be closely monitored. The community-lineage E. faecium CC94 clade was detected in patients with diarrhea, implying that community isolates might find their way into hospitals. Conclusion: This study highlights the One Health challenges posed by enterococci important to human health and the need to implement integrated preventive measures for their control.