Objective To establish an animal model of co-infection with Mycoplasma pneumoniae and influenza virus H1N1. The aim is to clarify the co-pathogenic mechanism of co-infection of the two pathogens and provide a theoretical basis for the clinical intervention of mixed respiratory tract infections.Methods Twenty-four Syrian hamsters were divided into four groups: control group, H1N1 influenza virus single-infection group, Mycoplasma pneumoniae single-infection group, and Mycoplasma pneumoniae and H1N1 influenza virus co-infection group. The animals were challenged via intranasal drops and tracheal injection. Clinical indicators were recorded for 14 days, physiological parameters and pathogen shedding were monitored, and animals were selected on days 7 and 14 post-infection for histopathological examination, viral antigen detection, and nucleic acid testing. Results Syrian hamsters in both single- and co-infected experimental groups exhibited clinical symptoms similar to human influenza or Mycoplasma pneumoniae infections. Nucleic acids of both pathogens were detected in nasal swabs, throat swabs, anal swabs, blood, and tissues. Histopathological results showed varying degrees of pathological damage in different tissues of the experimental groups. Conclusion The clinical symptoms, viral replication, and pathological manifestations confirm the successful establishment of a Syrian hamster model co-infected with Mycoplasma pneumoniae and influenza virus H1N1. This model provides an important experimental foundation for elucidating the mechanisms of respiratory co-infections, as well as for vaccine and drug development and clinical treatment strategies.