Creatine kinase (CK) system is known as a phosphoryl buffering and transfer system between mitochondria and ATPases. Functional coupling between CK and adenine nucleotide translocase, ATPases, and ATP-sensing potassium channel, has been demonstrated suggesting a major role of the CK system in the heart. To study CK system role, we used two lines of creatine-deficient mice, which lack either guanidinoacetate methyltransferase (GAMT) or arginine-glycine amidinotransferase (AGAT) on creatine synthesis pathway. Intriguingly, there have been no significant adaptations to the absence of active CK system in GAMT and AGAT mice cardiomyocytes from bioenergetics perspective: mitochondrial arrangement, respiration kinetics, activities of alternative pathway enzymes, intracellular compartmentation are the same in wild-type (WT) and knock-out (KO) littermates. Using whole-cell patch clamp, we studied how the absence of CK system influences calcium handling in cardiomyocytes of AGAT KO and WT littermates. Voltage dependence of L-type calcium channel (LTCC) current was significantly different in KO and WT animals with the significant differences between sexes within groups. LTCC amplitude was influenced by the absence of active CK system only in female mice in control conditions but was found to be the same in KO and WT male mice and mice of both sexes after adrenergic stimulation. The absence of active CK system had a major impact on calcium uptake, with the KO mice requiring more time for reducing calcium concentration to the resting level at the lower uptake rates. The effects of the active CK system absence on LTCC and calcium uptake, presumingly mainly by SR calcium ATPase, were fully reversed by creatine feeding to the KO animals. Our results suggest that there is a major role of the CK system in providing energy to excitation-contraction machinery in the heart to maintain normal calcium homeostasis.