Lactarius hatsudake is a common ectomycorrhizal edible mushroom in Pinus massoniana forests, and has important ecological and potential economic values. However, there are only a few reports on the establishment of Pinus massoniana–Lactarius hatsudake symbiosis. Here, we isolated a new strain of L. Lactarius hatsudake (GX01) from a local masson pine forest and established its ectomycorrhizal symbiosis with the P. massoniana. Potato dextrose agar (PDA) medium was optimal for the growth of L. hatsudake GX01. The saffron-to-brown ectomycorrhiza formed by L. hatsudake GX01 are usually bifurcated or coralloid shape, with a rod and a smooth surface, without emanating hyphae. The characteristic mantle and Hartig net structures of ectomycorrhizae were confirmed by microscope and scanning electron microscope (SEM). L. hatsudake GX01 can significantly promote the formation and development of lateral roots of P. massoniana seedlings during the early interaction. This study thus lays the foundation for subsequent study of the symbiotic molecular mechanism and application of P. massoniana–L. hatsudake symbiosis.