Two Ca²⁺ dependent signaling pathways, mediated by the Ca²⁺ activated phosphatase calcineurin and by the Ca²⁺ activated kinase Ca²⁺/calmodulin dependent kinase (CaMK), are both believed to function in fast-to-slow skeletal muscle fiber type transformation, but questions about the relative importance of the two pathways still remain. Here, the differential gene expression during fast-to-slow fiber type transformation was studied using cultured adult flexor digitorum brevis (FDB) fibers, and a custom mini microarray system, containing 21 fiber type-specific marker genes. After 3 days of culture, unstimulated fibers showed a generally slower gene expression profile. 3 days of electric-field stimulation of cultured FDB fibers with a slow-fiber-type pattern transformed the fibers to an even slower gene expression profile. Unstimulated FDB fibers overexpressing constitutively active calcineurin featured a slower gene expression profile, except 4 genes, indicating that transformation occurred, but was incomplete with activation of the calcineurin pathway alone. In both unstimulated FDB fibers and slow-type electrically stimulated FDB fibers, blocking of CaMK pathway with KN93 generated a faster gene expression profile, compared to the negative control KN92, indicating that CaMK pathway functions during the transformation induced by both unstimulated culturing and slow-fiber-type electrical stimulation. Moreover, neither the calcineurin nor the CaMK pathway alone could maximally activate the transformation, and coordination of the 2 pathways is required to accomplish a complete fast-to-slow fiber type transformation.