Negative temperature coefficient (NTC) thermistors are expected to be developed at cryogenic temperature sensing. In this paper, a kind of cryogenic thermistor was developed based on NTC response of single crystal ZnO microwires (MWs). The current–voltage (I–V) characterization demonstrated a NTC response, which was temperature dependence of resistance that decreased with increasing temperature. A sensitive NTC response, especially at the cryogenic temperature range, was observed. The defects in ZnO MWs play an important role in NTC response; therefore, we studied the origin of NTC effect associated with defects to improve the recognition of relationship between defects and the NTC effect and further optimize the temperature sensor design for high performance. Annealing at 800 °C in air was undertaken to make a significant influence on the concentration of defects in as-grown sample, and a series of temperature-dependent features were investigated by photoluminescence, Raman, XPS, and EPR measurements. The results indicated the zinc interstitials to be effective donors for sensitive NTC effect at the cryogenic region. This study provides insight into the ZnO NTC effect and sensitive cryogenic sensing technology.