The effect of Ni²⁺ on the growth and functional gene expression of the pure culture and co-culture of Acidithiobacillus thiooxidans and Leptospirillum ferriphilum has been studied. Compared with the pure culture, the co-culture showed a stronger sulfur and ferrous ion oxidation activity. At 100 mM, A. thiooxidans in co-culture grew faster and had 48 h shorter lag phases. The cell number of A. thiooxidans in co-culture was about 5 times higher than that in pure culture. The existence of A. thiooxidans in co-culture activated the expression of some metal resistance genes in L. ferriphilum at least 16 h in advance. A. thiooxidans in co-culture tends to chose more efficient pathways to transport nickel ion, ensuring the export of heavy metal was faster and more effective than that in pure culture. All the data indicated that there were synergetic interactions between iron- and sulfur-oxidizing bacteria under the stress of Ni²⁺.