Arbuscular mycorrhizal fungi (AMF) enhance plant drought tolerance partly by promoting soluble sugar accumulation, the mechanisms by which AMF colonization influences host sugar metabolism and associated gene expression under drought remain poorly understood. This study therefore examined the effects of inoculation with Funneliformis mosseae (T.H. Nicolson & Gerd.) C. Walker & A. Schüßler on the growth, water status, sugar profiles, and the expression/activity of sucrose-associated genes in the leaves of trifoliate orange (Citrus trifoliata L.) under water stress conditions. Over a 10-week water stress period, the root colonization by F. mosseae decreased by 14.36%, compared with ample water controls. Under water stress, F. mosseae inoculation significantly promoted plant growth performance (height, leaf number, and stem thickness), leaf water potential, nitrogen balance index, and chlorophyll index compared to those without inoculation. Ten sugar components (eight monosaccharides and two disaccharides) were detected. Inoculation with F. mosseae significantly elevated leaf contents of D-fructose, D-galactose, glucose, and inositol under both ample water and water stress conditions. It also increased leaf contents of D-arabinose under water stress, while decreasing the sucrose content under ample water conditions. The presence of the fungus boosted the acid invertase (AI) activity under water stress and up-regulated the relative expression of CtAI, CtNI, and CtSPS genes in leaves under both conditions. These gene expressions displayed a significantly positive correlation with root mycorrhizal colonization rate and glucose content, but a negative correlation with sucrose content. F. mosseae modulated leaf sugar profiles in trifoliate orange under water stress, particularly by regulating sucrose synthesis and cleavage through modulating the expression of sucrose-associated genes. Since this study focused on a single AMF species and leaf response, further work should investigate diverse AMF species and sugar profiles in other plant tissues under water stress.