Objective: This study aims to explore and compare guinea pig tuberculosis models using different doses of intranasal and aerosol infection routes, providing a foundation for establishing a standardized guinea pig tuberculosis model of respiratory tract infection. Methods: 24 female guinea pigs were randomly divided into 6 groups, with 4 guinea pigs in each group. The guinea pigs were infected with Mtb through either aerosol or intranasal at two doses respectively, and were equally divided into 3 groups (A/B/C, D/E/F). The general manifestations of the guinea pigs were observed after infection. All guinea pigs were dissected on the 14th day, and lung, spleen, and liver tissues were obtained for gross examination as well as histopathological analysis using HE staining to identify characteristic lesions associated with tuberculosis. Acid-fast staining was performed on in situ tissues and organs followed by bacterial culture to analyze bacterial load. Results: The guinea pigs in the four infection groups (B-C, E-F) exhibited macroscopic tuberculosis lesions in the lung, spleen, and liver. Histopathological examination revealed the presence of tuberculous granuloma lesions. Acid-fast staining and bacterial load analysis demonstrated that the bacteria were primarily localized in the lung tissue of the aerosol-infected group (B/C), with a few also present in the spleen and liver, and the bacterial load ranged is 10^4-5 CFU/ml. In the intranasal infection group (D/E), bacteria were found in the lung, spleen, and liver with a similar bacterial load of 10^4-5 CFU/ml. There was no significant difference observed in lesion severity or bacterial load between groups B-C and E-F; however, groups B, C, and F displayed low standard deviations for both pathology and etiology. Conclusion: A guinea pig model of acute tuberculosis has been successfully established using two different doses administered through distinct routes of infection. Pathological examination and pathogenic analysis demonstrated that an aerosol dose of 500 CFU effectively prepared a homogeneous model of acute tuberculosis with good consistency among subjects. Additionally, intranasal infection with 50,000 CFU Mtb also produced a relatively uniform model of tuberculosis; however, it should be noted that an aerosol infection at 500 CFU developed into an acute tuberculosis model more rapidly compared to intranasal infection at 50,000 CFU.