Abstract: The objective of this study was to determine the effects of applying single or multiple lactic acid bacteria (LAB) at ensiling on the fermentation quality and protein fractions of alfalfa (Medicago sativa). The protein fractions of alfalfa silage were calculated by Cornell net carbohydrate and protein system (CNCPS). The silage material was the first-cut alfalfa (425.6 g DM·kg-1 fresh herbage) harvested at budding stage. Forage was packed into 21 laboratory silos and ensiled for 40 days. The results showed that all treatments with LAB had significantly lower (P<0.05) concentration of NH3-N than untreated group, and butyric acid was not detected in these treatments. The treatment of single-strain (Lactobacillus plantarum) at 1×107 cfu·g-1 fresh herbage had the lowest concentration of NH3-N and the highest concentration of lactic acid. Silages treated with lactic acid bacteria (other than multi-strains at 1×105 cfu·g-1 fresh herbage) characterized by lower (P<0 05="" ph="" value="" there="" was="" no="" p="">0.05) treatment effect on the content of crude protein. The content of non-protein nitrogen (NPN) in silages with all additives of LAB was reduced when compared with untreated, and silages treated with compound LAB characterized by lower (P<0.05) NPN. Additives with single and multi-strains of LAB both resulted in alfalfa silage with low concentration of non-protein nitrogen (PA), high concentration of immediately degraded protein (PB1) and intermediately degraded protein (PB2). The concentration of true protein (PB) in silage with multi-strains of LAB was higher (P<0.05) than untreated. Therefore, high concentration of L. plantarum was beneficial to improve the fermentation quality of alfalfa, and the proteolysis, in some extent, could be inhibited by multiple LAB at ensiling.