Artificial pollination is a key technology with a wide range of applications in the fields of ecological research and agricultural practice. However, the effectiveness of pollination tools and pollen preservation has not received much attention. In this study, we investigated the effects of different artificial pollination methods and pollen storage conditions on the reproductive performance of Sagittaria trifolia. Results of two years showed that compared with direct contact pollination, pollination with brush tools significantly reduced fruiting probability and seed number in S. trifolia. The conditions of pollen handling also had significant effect on fruiting probability, in which half-hour field temperature storage had an advantage over control, whereas seed number per fruit and seed size remained stable. In summary, to aid artificial pollination in S. trifolia, pollination tools such as brushes should be avoided due to their negative effects on seed reproduction, and pollen in vitro can be preserved under field condition for an appropriate time. In related applications, researchers should be cautious of pollination tools and pay attention to the equivalence of different pollination methods prior to comparative experiments, in order to ensure scientific validity.