Herbaceous peony (Paeonia lactiflora Pall.) is a popular high-grade cut flower because of higher ornamental value. However, its short flowering time severely restricts the production and application of cut P. lactiflora flowers. In this study, nano-silver (NS) was applied to prolong the vase life of cut P. lactiflora flowers. Under the NS treatment, related physiological indices including relative electrical conductivity (REC), malondialdehyde (MDA), superoxide anion free radical (O2·-), hydrogen peroxide (H2O2) and free proline contents, and protective enzyme activities including superoxide dismutase (SOD), peroxidase (POD) and ascorbic acid peroxidase (APX) all increased in cut P. lactiflora flowers except soluble protein. Meanwhile, NS treatment increased relative water uptake (RWU) and Ag+ distribution. Moreover, the observation of microstructures indicated that the stem-ends without NS treatment were blocked by microbes which were identified as Alternaria sp. and Phoma sp., and NS effectively inhibited their growth by antibacterial efficacy observation. Additionally, three aquaporin genes (AQPs) including two plasma membrane intrinsic protein genes (PlPIP1;2, PlPIP2;1) and one NOD26-like intrinsic protein gene (PlNIP) were isolated, PlPIP1;2, and PlPIP2;1 that were induced by NS treatment took common effects on maintaining the water balance of cut P. lactiflora flowers. Consequently, the vase life of cut P. lactiflora flowers was prolonged and flower fresh weight together with flower diameter was well kept because of these above factors. These results would provide a theoretical basis for prolonging the vase life and improving the ornamental quality of cut P. lactiflora flowers with NS application.