The cadmium-resistant Cupriavidus sp. strain E324 has been previously shown to have a high potential for use in cadmium (Cd) remediation, due to its high capacity for cadmium bioaccumulation. According to the comparative genomic analysis, the strain E324 was most closely related to C. nantongensis X1T, indicating that the strain E324 should be re-identified as C. nantongensis. To unravel the Cd tolerance mechanisms of C. nantongensis strain E324, the transcriptional response of this strain to acute Cd exposure was assessed using RNA-seq-based transcriptome analysis, followed by validation through qRT-PCR. The results showed that the upregulated Differentially Expressed Genes (DEGs) were significantly enriched in categories related to metal binding and transport, phosphate transport, and oxidative stress response. Consistently, we observed significant increases in both the cell wall and intracellular contents of certain essential metals (Cu, Fe, Mn, and Zn) upon Cd exposure. Among these, only the Zn pretreatment resulting in high Zn accumulation in the cell walls could enhance bacterial growth under Cd stress conditions through its role in inhibiting Cd accumulation. Additionally, the promotion of catalase activity and glutathione metabolism upon Cd exposure to cope with Cd-induced oxidative stress was demonstrated. Meanwhile, the upregulation of phosphate transport-related genes upon Cd treatment seems to be the bacterial response to Cd-induced phosphate depletion. Altogether, our findings suggest that these adaptive responses are critical mechanisms contributing to increased Cd tolerance in C. nantongensis strain E324 via the enhancement of metal-chelating and antioxidant capacities of the cells.