Staphylococcus aureus possesses two distinct cardiolipin (CL) synthase genes, cls1 and cls2. It was previously shown that cls2 encodes a housekeeping-type CL synthase. However, the role of cls1 is elusive; a cls1 mutant was found to be equal to the wild type in terms of CL accumulation and stress tolerance. Here, we report that the physiological role of cls1 is to synthesize CL under conditions of acute low-pH stress. Below pH 2.6, the cls1 mutant (i.e. carrying Cls2 alone) could not produce CL, while the cls2 mutant (carrying Cls1) effectively accumulated CL. The cls1-dependent CL production was quick (within 5 min) and did not require de novo protein synthesis. Together with the results of phylogenetic analyses, our findings suggest that cls1 was generated through the duplication of cls2 after the divergence of the genus Staphylococcus and that the alternative CL synthase encoded by this gene confers improved survival in the face of acute acid stress.