The hypothesis that the Ajime-loach, Niwaella delicata, is guided to groundwater seepages by a positive thermotaxis in autumn, was tested by a field investigation and aquarium-based experiments. A total of 763 individuals of N. delicata were captured from October to November in a groundwater trap in the Yasu River, Shiga Prefecture. Niwaella delicata began to be captured as the temperature of the surface water fell to 15.8° ± 1.1°C (mean ± SD) and that of the groundwater to 15.5° ± 0.9°C. Groundwater was often warmer than surface water at night or occasionally all day, and the difference in temperature reached a maximum of 1.3°C at the night on 5 November. For the diel pattern of captures, nocturnal capture was higher than diurnal capture when the groundwater was warmer at night and colder during the daytime, whereas both diurnal and nocturnal captures were high when the groundwater was always warmer than the surface water. The aquarium-based experiments showed that N. delicata choose warmer water, ranging from 18.4° to 22.2°C, just before the capture period in the Yasu River, and are sensitive to differences in water temperature of 1.3° ± 0.1°C. Although the present results broadly support the hypothesis, a part of the results indicates that water temperature gradients may not be the only factor involved in the groundwater selection of N. delicata.