Potassium plays important roles in most plant physiological processes. Arbuscular mycorrhizal (AM) fungi promote plant water and mineral nutrient acquisition to promote plant growth. However, few studies have focused on the effect of AM colonization on potassium uptake by the host plant. In this study, the effects of an AM fungus (Rhizophagus irregularis) and potassium concentration (0, 3, or 10 mM K+) onLycium barbarumwere evaluated. A split-root test withL. barbarumseedlings was conducted, and the potassium uptake capacity of LbKAT3 was verified in yeast. A tobacco line overexpressingLbKAT3was generated and mycorrhizal functions under two potassium concentrations (0.2 and 2 mM K+) were studied. Inoculation ofR. irregularisand application of potassium increased the dry weight, and potassium and phosphorus contents ofL. barbarum, and increased the colonization rate and arbuscule abundance ofR. irregularis. In addition, the expression ofLbKAT3andAQPgenes inL. barbarumwas upregulated. Inoculation ofR. irregularisinducedLbPT4,Rir-AQP1, andRir-AQP2expression, and application of potassium upregulated the expression of these genes. Inoculation with the AM fungus locally regulated the expression ofLbKAT3. Inoculation ofR. irregularisimproved the growth, and potassium and phosphorus contents, and inducedNtPT4,Rir-AQP1, andRir-AQP2expression in tobacco overexpressingLbKAT3under both potassium concentrations. Overexpression ofLbKAT3in tobacco improved the growth, potassium accumulation, and AM colonization, and upregulated the expression ofNtPT4andRir-AQP1in mycorrhizal tobacco. The results suggest thatLbKAT3may assist in mycorrhizal potassium uptake, and overexpression ofLbKAT3may promote potassium, phosphorus, and water transport from the AM fungus to tobacco.