The Red Queen hypothesis depicts evolution as the continual struggle to adapt. According to this hypothesis, new genes, especially those originating from nongenic sequences (i.e., de novo genes), are eliminated unless they evolve continually in adaptation to a changing environment. Here, we analyze two Drosophila de novo miRNAs that are expressed in a testis-specific manner with very high rates of evolution in their DNA sequence. We knocked out these miRNAs in two sibling species and investigated their contributions to different fitness components. We observed that the fitness contributions of miR-975 in Drosophila simulans seem positive, in contrast to its neutral contributions in D. melanogaster, whereas miR-983 appears to have negative contributions in both species, as the fitness of the knockout mutant increases. As predicted by the Red Queen hypothesis, the fitness difference of these de novo miRNAs indicates their different fates.