The objective of present study was to investigate the changes of total fatty acid (FA) content and fatty acid composition in high moisture alfalfa (DM=229g/kg fresh weight) silage treated without (Control) or with Lactobacillus buchneri (LB), L. plantarum (LP), propionic acid additive (PA) and tea polyphenols (TP) during ensiling and after exposure to air. A 750g of alfalfa material was filled into the experimental silo (polyvinyl chloride bottle, 1000mL capacity). After ensiling for 65days at room temperature, a part of silages (200g) was sampled and the others (450g) were exposed to air for 6days. Butyric acid (>30g/kg DM) was more than lactic acid (<1g/kg DM) in the control and TP silages due to the low contents of DM and water-soluble carbohydrates, and high buffer capacity, indicating poor fermentation quality. LB, LP, and PA promoted lactic fermentation, restrained butyric fermentation and thus improved fermentation quality of silages. Unsaturated fatty acids (UFA) lipolysis and saturated fatty acids (SFA) synthesis occurred in the control silage during ensiling, mainly the lipolysis of C18:2n-6 and C18:3n-3 and synthesis of C6:0. Compared with the control, LB, LP, and PA restrained butyric fermentation and synthesis of C6:0, meanwhile LB and LP did not relieve the lipolysis of UFA while PA relieved it; TP relieved synthesis of C6:0 and had a slight lipolysis of UFA. After exposure to air for 6days, the control, LB, PA, and TP silages had better aerobic stability than LP silage, supported by higher temperature in LP silage than in the control, LB, PA, and TP silages. Like volatile FA, most of the FA composition had a positive correlation with aerobic stability. PA silage had the highest total FA content (33.8g/kg DM), the proportions of UFA, C18:2n6 and C18:3n3 due to its lower pH (<4.60) and amounts of aerobic bacteria (<5.0lgcfu/g FM) and yeasts (<2.6lgcfu/g FM) than the control, LB and LP silages, and its lower pH and aerobic bacteria number than TP silage. Therefore, PA kept silage aerobic stability and was a good additive to conserve FA of silage after exposure to air for 6days.