MICU1 and MICU2 are the main regulators of the mitochondrial Ca^2 +-uniporter (MCU), but their precise functional role is still under debate. We show here that MICU2 behaves as a pure inhibitor of MCU at low cytosolic [Ca^2 +] ([Ca^2 +]_c), though its effects decrease as [Ca^2 +]_c is increased and disappear above 7 μM. Regarding MICU1, studying its effects is more difficult because knockdown of MICU1 leads also to loss of MICU2. However, while knockdown of MICU2 induces only a persistent increase in mitochondrial Ca^2 + uptake, knockdown of MICU1 also induces a peculiar use-dependent transient activation of MCU that cannot be attributed to the parallel loss of MICU2. Therefore, MICU1 is endowed with a specific inhibitory effect on MCU at low [Ca^2 +]_c, separate and kinetically different from that of MICU2. On the other hand, we and others have shown previously that MICU1 activates MCU at [Ca^2 +]_c above 2.5 μM. Thus, MICU1 has a double role in MCU regulation, inhibitory at low [Ca^2 +]_c and activatory at high [Ca^2 +]_c.