Acidithiobacillus ferrooxidans ATCC 23270 is an important model organism for bioleaching and bioremediation studies owing to its diverse metabolic capabilities, whereas lix984n is a widely used extractant. Little is known about the response of cbb genes in A. ferrooxidans to lix984n shock. Thus, to elucidate the response of the CO2 fixation genes in A. ferrooxidans ATCC 23270 to the addition of lix984n, the gene expression of cbb genes was examined using a real-time PCR. Although a natural increase or decrease in the expression of most cbb genes was observed after 5 min of shock with 3% (v/v) lix984n, sdhC and cbbR exhibited quick responses to the shock. Ten min of shock had a greater effect on the cbb gene expression, yet 15 min of shock had a significant effect on the Calvin cycle in A. ferrooxidans ATCC 23270, as the expression of all the cbb genes reached a very high level. Therefore, after a short lix984n shock, a solution of A. ferrooxidans can be re-used for bioleaching.