Niche separation may be the key to promoting the long-term coexistence of introduced and native species. Physical alterations to the environment (habitat manipulation) or re-introducing native species to former habitats can exploit the maladapted traits of introduced species to create a refuge for native species. No two species have identical niches because evolutionary constraints differ between species with different evolutionary histories. Our objectives were to determine if cold temperatures could promote coexistence between native least chub and introduced western mosquitofish. We used individual scale and population scale experiments to test four hypotheses: 1) colder temperatures would reduce the aggressive behavior and predatory effects of western mosquitofish on least chub, 2) colder temperatures would reduce the effect of western mosquitofish on the habitat use, activity, and feeding of least chub, 3) western mosquitofish would not be able to overwinter without warm refuges, and 4) western mosquitofish reproduction would be delayed or absent at colder temperatures, whereas colder temperatures would not inhibit least chub recruitment. At the individual scale cold temperatures reduced the aggression and predation of western mosquitofish on least chub. However at the population scale there was little recruitment in the cold treatment and juvenile least chub did not survive the winter in the cold treatment. Adult least chub successfully overwintered at freezing temperatures whereas western mosquitofish had no recruitment in the cold treatment during the summer and no western mosquitofish survived the winter. There is adequate niche separation among the adults to promote coexistence but the juveniles of both species require warm habitat in the spring and summer to survive freezing winter temperatures. Habitat manipulation may reduce the availability of warm winter refuges for western mosquitofish while leaving warm habitats during the spring for least chub spawning and recruitment. Transplanting least chub to former cold habitats could eliminate western mosquitofish because of niche separation between the species along a temperature gradient. We suggest that the niche separation hypothesis has general application for the restoration of a variety of threatened native species.