Pathological cardiac hypertrophy (PCH) is associated with the development of arrhythmia and congestive heart failure. While calcium (Ca²⁺) is implicated in hypertrophic signaling pathways, the specific role of Ca²⁺ influx through the L-type Ca²⁺ channel (I_Ca-L) has been controversial and is the topic of this study. To determine if and how sustained increases in I_Ca-L induce PCH, transgenic mouse models with low (LE) and high (HE) expression levels of the β2a subunit of Ca²⁺ channels (β2a) and in cultured adult feline (AF) and neonatal rat (NR) ventricular myocytes (VMs) infected with an adenovirus containing a β2a-GFP were used. In vivo, β2a LE and HE mice had increased heart weight to body weight ratio, posterior wall and interventricular septal thickness, tissue fibrosis, myocyte volume, and cross-sectional area and the expression of PCH markers in a time- and dose-dependent manner. PCH was associated with a hypercontractile phenotype including enhanced I_Ca-L, fractional shortening, peak Ca²⁺ transient, at the myocyte level, greater ejection fraction, and fractional shortening at the organ level. In addition, LE mice had an exaggerated hypertrophic response to transverse aortic constriction. In vitro overexpression of β2a in cultured AFVMs increased I_Ca-L, cell volume, protein synthesis, NFAT, and HDAC translocations and in NRVMs increased surface area. These effects were abolished by the blockade of I_Ca-L, intracellular Ca²⁺, calcineurin, CaMKII, and SERCA. In conclusion, increasing I_Ca-L is sufficient to induce PCH through the calcineurin/NFAT and CaMKII/HDAC pathways. Both cytosolic and SR/ER-nuclear envelop Ca²⁺ pools were shown to be involved.