Generating, manipulating and detecting electron spin polarization and coherence at room temperature is at the heart of future spintronics and spin-based quantum information technology(1-4). Spin filtering, which is a key issue for spintronic applications, has been demonstrated by using ferromagnetic metals(5-8), diluted magnetic semiconductors(9,10), quantum point contacts(11), quantum dots(12), carbon nanotubes(13), multiferroics(14) and so on. This filtering effect was so far restricted to a limited efficiency and primarily at low temperatures or under a magnetic field. Here, we provide direct and unambiguous experimental proof that an electron-spin-polarized defect, such as a Ga(i) self-interstitial in dilute nitride GaNAs, can effectively deplete conduction electrons with an opposite spin orientation and can thus turn the non-magnetic semiconductor into an efficient spin filter operating at room temperature and zero magnetic field. This work shows the potential of such defect-engineered, switchable spin filters as an attractive alternative to generate, amplify and detect electron spin polarization at room temperature without a magnetic material or external magnetic fields.