Cooling can induce Ca2+ signaling via activation of temperature-sensitive ion channels such as TRPM8, TRPA1 and ryanodine receptor channels. Here we have studied the mechanism of cooling-evoked Ca2+ signaling in mouse olfactory ensheathing cells (OECs), a specialized type of glial cells in the olfactory nerve layer of the olfactory bulb. Reducing the temperature from above 30°C to 28°C and below triggered Ca2+ transients that persisted in the absence of external Ca2+, but were suppressed after Ca2+ store depletion by cyclopiazonic acid. Cooling-evoked Ca2+ transients were present in mice deficient of TRPM8 and TRPA1, and were not inhibited by ryanodine receptor antagonists. Inhibition of InsP3 receptors with 2-APB and caffeine entirely blocked cooling-evoked Ca2+ transients. Moderate Ca2+ increases, as evoked by flash photolysis of NP-EGTA (caged Ca2+) and cyclopiazonic acid, triggered InsP3 receptor-mediated Ca2+ release at 22°C, but not at 31°C. The results suggest that InsP3 receptors mediate Ca2+-induced Ca2+ release in OECs, and that this Ca2+ release is temperature-sensitive and can be suppressed at temperatures above 28°C.