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The Chinese soft-shelled turtle (Pelodiscus sinensis) is an important freshwater aquaculture turtle due to its taste and nutritional and medicinal value. More ecological culturing modes, such as rice–turtle co-culture, should be developed to meet the ecological benefit demand. We compared growth, physiological parameters, and transcriptome data to detect the physiological responses and regulatory mechanisms of pond-cultured turtles as compared to co-cultured turtles. The co-cultured turtles grew slower than pond-cultured turtles. The gonadosomatic index of co-cultured male turtles was lower than that of pond-cultured male turtles, and both the mesenteric fat index and limb fat index were lower in co-cultured turtles than in pond-cultured turtles (p < 0.05). The blood GLU of the co-cultured turtles was significantly lower than the GLU of the pond-cultured turtles (p < 0.05), while the values of CRE, UA, BUN, AKP, ACP, GOT, and CAT were higher in the co-cultured turtles than in the pond-cultured turtles (p < 0.05). In total, 246 and 598 differentially expressed genes (DEGs) were identified in the brain and gut from turtles cultured in the two different modes, respectively. More DEGs were related to environmental information processing, metabolism, and human diseases. In the brain, the top enriched pathways of DEGs included the longevity regulating pathway, glycerolipid metabolism, cytokine–cytokine receptor interaction, Toll-like receptor signaling pathway, and PI3K-Akt signaling pathway, while in the gut, the top enriched pathways of DEGs included the cell cycle, DNA replication, cellular senescence, and p53 signaling pathway. The turtles acclimated to the different culturing conditions by adjusting their growth, physiological, and biochemical characteristics and related gene expression during a short culture period.