Abstract: This study aims to clarify the role of arbuscular mycorrhizal fungi (AMF) in the growth and physiological characteristics of flue-cured tobacco seedlings. A pot experiment was conducted to detect the impacts of three different inoculation methods and five AMF strains (Acaulospora scrobiculata, As; Rhizophagus intraradices, Ri; Funneliformis mosseae, Fm; Claroideoglomus etunicatum, Ce; Glomus versiforme, Gv) on the growth parameters, photosynthetic performance, carbon and nitrogen metabolism, and mineral nutrient uptake of the flue-cured tobacco variety K326 seedlings. Results showed that the five AMF strains established symbiotic relationships with the tobacco roots under the three inoculation methods. Compared to the non-inoculated control, AMF inoculation significantly increased the root projection area, total root length, root tip number, dry matter accumulation, and net photosynthetic rate of tobacco plants. In particular, under the inoculation method three (where the inoculum was placed approximately 0.5 cm away from the tobacco roots), the tobacco seedlings inoculated with Acaulospora scrobiculata (As) exhibited significantly higher total root length, root projection area, dry matter accumulation, and net photosynthetic rate than those inoculated with other AMF strains. Furthermore, under the method three, the seedlings inoculated with As displayed significantly higher level of root diameter, dry matter accumulation, net photosynthetic rate, sucrose phosphate synthase activity, and nitrogen content in the underground parts, compared to those inoculated under the method two (where the inoculum was in direct contact with the roots). These findings suggest that AMF inoculation can effectively promote the root and leaf development of flue-cured tobacco seedlings, and enhance its nutrient uptake. The As strain, when inoculated under the method three, forms the most beneficial symbiotic relationship, yielding superior results.