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Background In water lily (Nymphaea) hybrid breeding, breeders often encounter non-viable seeds, which make it difficult to transfer desired or targeted genes of different Nymphaea germplasm. We found that pre-fertilization barriers were the main factor in the failure of the hybridization of Nymphaea. The mechanism of low compatibility between the pollen and stigma remains unclear; therefore, we studied the differences of stigma transcripts and proteomes at 0, 2, and 6 hours after pollination (HAP). Moreover, some regulatory genes and functional proteins that may cause pre-fertilization barriers in Nymphaea were identified. Results RNA-seq was performed for three comparisons (2 vs 0 HAP, 6 vs 2 HAP, 6 vs 0 HAP), and the number of differentially expressed genes (DEGs) was 8,789 (4,680 were up-regulated), 6,401 (3,020 were up-regulated), and 11,284 (6,148 were up-regulated), respectively. Using label-free analysis, 75 (2 vs 0 HAP) proteins (43 increased and 32 decreased), nine (6 vs 2 HAP) proteins (three increased and six decreased), and 90 (6 vs 0 HAP) proteins (52 increased and 38 decreased) were defined as differentially expressed proteins (DEPs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the DEGs and DEPs were mainly involved in cell wall organization or biogenesis, S-adenosylmethionine (SAM) metabolism, hydrogen peroxide decomposition and metabolism, reactive oxygen species (ROS) metabolism, secondary metabolism, secondary metabolite biosynthesis, and phenylpropanoid biosynthesis. Conclusions These results indicated that genes involved in regulation and gene pathways involved in stress response might overlap in the process of pollination process forming the hybridization barrier in Nymphaea. These results indicate that the pollen tube entering stigma tissue was very similar to the process of external injury, further suggesting that these genes involved in defense and stress response were necessary in the early stage of interaction between the pollen and stigma. This study strengthens our understanding of the mechanism of pre-fertilization barriers in Nymphaea at the molecular level, and provides a theoretical basis for overcoming the pre-fertilization barriers in Nymphaea in the future.