Polyamine oxidases (PAOs) are key enzymes in polyamine metabolism and are related to the tolerance of plants to abiotic stresses. In this study, overexpression of cucumber (Cucumis sativus L.) PAO2 (CsPAO2) in Arabidopsis resulted in increased activity of the antioxidant enzyme and accelerated conversion from Put to Spd and Spm, while malondialdehyde content (MDA) and electrolyte leakage (EL) was decreased when compared with wild type, leading to enhanced plant growth under salt stress. Photosystem Ⅰ assembly 3 in cucumber (CsPSA3) was revealed as an interacting protein of CsPAO2 by screening yeast two-hybrid library combined with in vitro and in vivo methods. Then, CsPAO2 and CsPSA3 were silenced in cucumber via virus-mediated gene silencing (VIGS) with pV190 as the empty vector. Under salt stress, net photosynthetic rate (Pn) and transpiration rate (Tr) of CsPAO2-silencing plants were lower than pV190-silencing plants, and EL in root was higher than pV190-silencing plants, indicating that CsPAO2-silencing plants suffered more serious salt stress damage. However, photosynthetic parameters of CsPSA3-silencing plants were all higher than those of CsPAO2 and pV190-silencing plants, thereby enhancing the photosynthesis process. Moreover, CsPSA3 silencing reduced the EL in both leaves and roots when compared with CsPAO2-silencing plants, but the EL only in leaves was significantly lower than the other two gene-silencing plants, and conversion from Put to Spd and Spm in leaf was also promoted, suggesting that CsPSA3 interacts with CsPAO2 in leaves to participate in the regulation of salt tolerance through photosynthesis and polyamine conversion.