We present the response of a NaI(Tl) crystal in terms of the light yield and pulse shape characteristics at two different temperatures: 22 °C (room temperature) and −35 °C (low temperature). The light yield is measured using 59.54keV γ-rays from a 241Am source relative to the mean charge of single photoelectrons. At the low temperature, we measure a 4.7 ± 1.3% increase in the light yield compared to that at room temperature. A significantly increased decay time is also observed at the low temperature. The responses to nuclear recoil events are measured using neutrons from a 252Cf source and compared to those to electron recoil events. The measured pulse shape discrimination (PSD) power of the NaI(Tl) crystal at the low temperature is found to be improved in the entire energy range studied because of the increased light yield and the different scintillation characteristics. We also find a 9.23 ± 0.26% increased quenching factor of α-induced events, which is the light yield ratio of α recoil to electron recoil, at the low temperature. This supports the possibility of an increased quenching factor of the nuclear recoil events that are known to have similar processes of dark matter interaction. The increased light yield and the improved PSD power of nuclear recoil events enhance the sensitivity for dark matter detection via dark matter–nuclei interactions.