To improve our understanding of the timing of cold stress and its effect on Pythium damping-off, we performed a factorial experiment with two cold stress temperatures (4 and 10°C); exposure to 96 h of cold stress at 0, 1, 2, 4, 6, and 8 days after planting; and inoculation with Pythium sylvaticum-infested millet or control. Increased susceptibility to damping-off resulting in reduced emergence was found in inoculated plants when the cold stress period began 2 or 4 days after planting. In the noninoculated controls, no effect of cold stress on emergence was observed. Slower seedling growth was observed during the cold stress period and in inoculated plants after exposure to cold stress. Seed exudation, mycelial growth, and sporangia germination of P. sylvaticum was evaluated at 4, 10, and 18°C. The greatest seed exudation was observed at 4°C. Low temperatures delayed mycelial growth of P. sylvaticum, although the pathogen was still able to grow at 4°C. Sporangia incubated for 3 h at 18°C in the presence of seed exudates had higher germination in comparison with sporangia incubated at 10 or 4°C. Moreover, more sporangia germinated in response to seed exudates that were previously collected from seed imbibed for 24 h at low temperatures (4°C). These results suggest that cold stress 2 to 4 days after planting increases soybean susceptibility to damping-off, presumably because of increased seed exudation and delayed seedling growth.