It has been proposed that the highly homologous and pertussis toxin (PTX)-sensitive cardiac Gαi isoforms, Gαi2 and Gαi3, are functionally distinct. Moreover, they are upregulated in heart failure. However, their stimulation by the parasympathic and sympathic nervous system and their effects on cardiac L-type calcium channel regulation remain unclear. In this study, the Gαi isoform-specific modulation of L- type calcium channels at the whole-cell level is investigated, using cardiomyocytes from mice lacking either Gαi2 (Gαi2-/-) or Gαi3 (Gαi3-/-) protein. In these cardiomyocytes, the other Gαi isoform is enhanced on mRNA and protein levels, whereas channel expression levels of Cavα and Cavβ subunits are nearly unchanged. Furthermore, subtype-specific signalling effects are detected. In particular, calcium current density in Gαi2-/- cardiomyocytes is significantly decreased, whereas it is significantly increased in Gαi3-/- cells as compared to control animals. Interestingly, only genetic or PTX-mediated abolition of Gαi2 function causes a steady-state inactivation shift towards hyperpolarized potentials and a delayed calcium current recovery. This novel Gαi2-associated effect is reflected by alterations in calcium channel kinetics and likely involves activation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signalling pathways