Negative interspecific mating interactions, known as reproductive interference, can hamper species coexistence in a local patch and promote niche partitioning or geographical segregation of closely related species. Conspecific sperm precedence (CSP), which occurs when females that have mated with both conspecific and heterospecific males preferentially use conspecific sperm for fertilization, might contribute to species coexistence by mitigating the costs of interspecific mating and hybridization. We discussed whether two species exhibiting CSP can coexist in a local environment in the presence of reproductive interference. First, using a behaviorally explicit mathematical model, we demonstrated that two species characterized by negative mating interactions are unlikely to coexist because the costs of reproductive interference, such as loss of mating opportunity with conspecific partners, are inevitably incurred when individuals of both species are present. Second, we experimentally examined differences in mating activity and preference in two Harmonia ladybird species known to exhibit CSP. These behavioral differences may lead to local extinction of H. yedoensis because of reproductive interference by H. axyridis. This prediction is consistent with field observations that H. axyridis uses various food sources and habitats whereas H. yedoensis is confined to a less preferred prey item and a pine tree habitat. Finally, by a comparative approach, we observed that niche partitioning or parapatric distribution, but not sympatric coexistence in the same habitat, is maintained between species with CSP belonging to a wide range of taxa, including vertebrates and invertebrates living in aquatic or terrestrial environments. Taken together, it is possible that reproductive interference may destabilize local coexistence even in closely related species that exhibit CSP.