Magnolia macclurei (Dandy) Figlar is a fast-growing hardwood species indigenous to northern Vietnam and southern China. As many species in the family Magnoliaceae, the shoot proliferation and in vitro rooting of this species are challenging. In this study the effects of light-emitting diode (LED) sources [blue (BB), yellow (YY), and red (RR) LEDs] and of fluorescence lamp (white, FLW) on in vitro shoot proliferation and rooting of M. macclurei, were researched and the physiological responses of plantlets were evaluated by checking the nutrient status and the pigment composition. Moreover, the tissue culture efficiency was scored under the different light regimes. Compared with FLW, YY, and RR LEDs significantly promoted the shoot growth and biomass accumulation, while BB LED had inhibiting effect. YY LED resulted in much smaller leaves and slightly fewer necrotic leaves than RR LED. The nutrient contents in both foliage and stem were obviously the lowest under RR LED, and were higher under YY LED and FLW. The chlorophyll contents, and the ratio of chlorophyll a to carotenoid were the highest under YY LED. The rooting rates under YY and RR LEDs were all above 80%, much higher than those under FLW and BB LED. YY LED was thus recommended for application in the practice of tissue culture for this species.