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<i>Vibrio parahaemolyticus</i> is the primary foodborne pathogen known to cause gastrointestinal infections in humans. Nevertheless, the molecular mechanisms of <i>V. parahaemolyticus</i> pathogenicity are not fully understood. Prophages carry virulence and antibiotic resistance genes commonly found in <i>Vibrio</i> populations, and they facilitate the spread of virulence and the emergence of pathogenic <i>Vibrio</i> strains. In this study, we characterized three such genes, <i>VpaChn25_0713</i>, <i>VpaChn25_0714</i>, and <i>VpaChn25_RS25055</i>, within the largest prophage gene cluster in <i>V. parahaemolyticus</i> CHN25. The deletion mutants Δ<i>VpaChn25_RS25055</i>, Δ<i>VpaChn25_0713</i>, Δ<i>VpaChn25_0714</i>, and Δ<i>VpaChn25_RS25055-0713-0714</i> were derived with homologous recombination, and the complementary mutants Δ<i>VpaChn25_0713</i>-com, Δ<i>VpaChn25_0714</i>-com, Δ<i>VpaChn25_RS25055</i>-com, Δ<i>VpaChn25_RS25055-0713-0714</i>-com were also constructed. In the absence of the <i>VpaChn25_RS25055</i>, <i>VpaChn25_0713</i>, <i>VpaChn25_0714</i>, and <i>VpaChn25_RS25055-0713-0714</i> genes, the mutants showed significant reductions in low-temperature survivability and biofilm formation (<i>p</i> < 0.001). The Δ<i>VpaChn25_0713</i>, Δ<i>VpaChn25_RS25055</i>, and Δ<i>VpaChn25_RS25055-0713-0714</i> mutants were also significantly defective in swimming motility (<i>p</i> < 0.001). In the Caco-2 model, the above four mutants attenuated the cytotoxic effects of <i>V. parahaemolyticus</i> CHN25 on human intestinal epithelial cells (<i>p</i> < 0.01), especially the Δ<i>VpaChn25_RS25055</i> and Δ<i>VpaChn25_RS25055-0713-0714</i> mutants. Transcriptomic analysis showed that 15, 14, 8, and 11 metabolic pathways were changed in the Δ<i>VpaChn25_RS25055</i>, Δ<i>VpaChn25_0713</i>, Δ<i>VpaChn25_0714</i>, and Δ<i>VpaChn25_RS25055-0713-0714</i> mutants, respectively. We labeled the <i>VpaChn25_RS25055</i> gene with superfolder green fluorescent protein (sfGFP) and found it localized at both poles of the bacteria cell. In addition, we analyzed the evolutionary origins of the above genes. In summary, the prophage genes <i>VpaChn25_0713</i>, <i>VpaChn25_0714</i>, and <i>VpaChn25_RS25055</i> enhance <i>V. parahaemolyticus</i> CHN25’s survival in the environment and host. Our work improves the comprehension of the synergy between prophage-associated genes and the evolutionary process of <i>V. parahaemolyticus</i>.