Arbuscular mycorrhizal fungi (AMF) can benefit many plants, but their effects on walnuts are not yet known. The present study aimed to analyze the effect of five AMF species, namely, Acaulospora scrobiculata, Diversispora spurca, Glomus etunicatum, G. mosseae and G. versiforme on biomass production, chlorophyll contents, sugar fraction contents, and mineral element contents of walnut (Juglans regia L.) seedlings. The five AMF species colonized roots of walnut, established mycorrhizas in roots and hyphae in soil, and released easily extractable glomalin-related soil protein into soil, whilst D. spurca exhibited the best effect. All the AMF inoculations, except A. scrobiculata, stimulated shoot and root biomass production. Mycorrhizal fungal inoculations collectively increased leaf chlorophyll a, chlorophyll b, and total chlorophyll a+b concentrations, and thus promoted leaf sucrose accumulation, which provides an important mycorrhiza-carbon source to roots. AMF inoculations conferred a positive effect on leaf N, P, K, Mg, Fe, B, Zn and Cu contents, while they reduced leaf Mn contents. These results concluded that AMF were beneficial to the growth and physiological activities of walnut, which gives the support for the AMF application in walnut.