Anoctamin-6 (ANO6) belongs to a family of calcium (Ca2+)-activated chloride channels (CaCCs), with three types of splicing variants (V1, V2, and V5) showing plasma membrane expression1. Unlike other CaCCs, ANO6 requires a non-physiological intracellular free calcium concentration ([Ca2+]i > 1 μM), and needs several minutes for full activation under a whole-cell patch clamp2. Therefore, the physiological role of ANO6 as an ion channel is in doubt; it is more commonly considered as a Ca2+-dependent phospholipid scramblase3. Here, we demonstrated that physiological temperature (37°C) increased the Ca2+ sensitivity of ANO6 under a whole-cell patch clamp; V1 was activated by 1 μM [Ca2+]i, whereas V2 and V5 were activated by 300 nM [Ca2+]i. Upon temperature increase to 42°C, all ANO6 variants can be activated by 100 nM [Ca2+]i. Moreover, 37°C condition significantly accelerated the delayed time to full activation the three variants. Notably, the temperature-dependent Ca2+-sensitisation of ANO6 became insignificant under inside-out path clamp, suggesting a critical role of unknown cytosolic factors. Unlike the channel activity, physiological temperature did not induce the scramblase function of ANO6 with submicromolar [Ca2+]i (300 nM), irrespective of variant type. Our results reveal the physiologically meaningful anion conducting property of ANO6, which might precede the scramblase activity.