The calcium-activated phosphatase calcineurin is regulated by a binding cofactor known as modulatory calcineurin-interacting protein (MCIP) in yeast up through mammals. The physiologic function of MCIP remains an area of ongoing investigation, because both positive and negative calcineurin regulatory effects have been reported. Here we disrupted the mcip1 and mcip2 genes in the mouse and provide multiple lines of evidence that endogenous MCIP functions as a calcineurin facilitator in vivo. Mouse embryonic fibroblasts deficient in both mcip1/2 showed impaired activation of nuclear factor of activated T cells (NFAT), suggesting that MCIP is required for efficient calcineurin-NFAT coupling. Mice deficient in mcip1/2 showed a dramatic impairment in cardiac hypertrophy induced by pressure overload, neuroendocrine stimulation, or exercise, similar to mice lacking calcineurin Abeta. Moreover, simultaneous deletion of calcineurin Abeta in the mcip1/2-null background did not rescue impaired hypertrophic growth after pressure overload. Slow/oxidative fiber-type switching in skeletal muscle after exercise stimulation was also impaired in mcip1/2 mice, similar to calcineurin Abeta-null mice. Moreover, CD4(+) T cells from mcip1/2-null mice showed enhanced apoptosis that was further increased by loss of calcineurin Abeta. Finally, mcip1/2-null mice displayed a neurologic phenotype that was similar to calcineurin Abeta-null mice, such as increased locomotor activity and impaired working memory. Thus, a loss-of-function analysis suggests that MCIPs serve either a permissive or facilitative function for calcineurin-NFAT signaling in vivo.