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. Spin filtering, which is a key issue for spintronic applications, has been demonstrated by using ferromagnetic metals, diluted magnetic semiconductors, quantum point contacts, quantum dots, carbon nanotubes, multiferroics 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.