The proliferation mechanism of Lactobacillus plantarum RB1 promoted by stachyose was investigated in this work. The hydrolysis of stachyose, the glycometabolism, and the cytoactivity of L. plantarum RB1 were detected after proliferation. The specific activity of alpha-galactosidase of L. plantarum RB1 in the stachyose group was significantly higher than the control group (without stachyose), which indicated that the stachyose induced L. plantarum RB1 and produced more alpha-galactosidase to hydrolyze stachyose. The glycometabolism which includes glycolysis and tricarboxylic acid (TCA) cycle was significantly enhanced in the stachyose group compared with the control group. For the glycolysis, the reducing sugar content in the fermentation broth was significantly lower, while the lactic acid content and the specific activity of lactic dehydrogenase (LDH) as the key enzyme in glycolysis were higher than in the control group. For the TCA cycle, the specific activity of pyruvate dehydrogenase (PDH) as a gatekeeping enzyme leads glycolysis to TCA cycle energy-generating pathways was significantly enhanced compared with the control group. Moreover, the cell metabolic activity of L. plantarum RB1 in stachyose was significantly higher than the control group. These results indicated that the stachyose highly promotes proliferation of lactic acid bacteria (LAB) by inducing LAB to produce more alpha-galactosidase to hydrolyze stachyose, increasing glycometabolism and cytoactivity of LAB, which revealed the mechanisms how the stachyose promotes the proliferation of LAB.