Background The multi-meric calcium/calmodulin-dependent protein kinase II (CaMKII) is the main CaMK in skeletal muscle and its expression increases with endurance training. CaMK family members are implicated in contraction-induced regulation of calcium handling, fast myosin type IIA expression and mitochondrial biogenesis. The objective of this study was to investigate the role of an increased CaMKII content for the expression of the contractile and mitochondrial phenotype in vivo. Towards this end we attempted to co-express alpha- and beta-CaMKII isoforms in skeletal muscle and characterised the effect on the contractile and mitochondrial phenotype. Results Fast-twitch muscle m. gastrocnemius (GM) and slow-twitch muscle m. soleus (SOL) of the right leg of 3-month old rats were transfected via electro-transfer of injected expression plasmids for native α/β CaMKII. Effects were identified from the comparison to control-transfected muscles of the contralateral leg and non-transfected muscles. α/β CaMKII content in muscle fibres was 4-5-fold increased 7 days after transfection. The transfection rate was more pronounced in SOL than GM muscle (i.e. 12.6 vs. 3.5%). The overexpressed α/β CaMKII was functional as shown through increased threonine 287 phosphorylation of β-CaMKII after isometric exercise and down-regulated transcripts COXI, COXIV, SDHB after high-intensity exercise in situ. α/β CaMKII overexpression under normal cage activity accelerated excitation-contraction coupling and relaxation in SOL muscle in association with increased SERCA2, ANXV and fast myosin type IIA/X content but did not affect mitochondrial protein content. These effects were observed on a background of regenerating muscle fibres. Conclusion Elevated CaMKII content promotes a slow-to-fast type fibre shift in regenerating muscle but is not sufficient to stimulate mitochondrial biogenesis in the absence of an endurance stimulus.