The 95kDa triadin (Trisk 95), the main isoform in skeletal muscle sarcoplasmic reticulum, interacts with both the ryanodin receptor and a calsequestrin. Earlier reports revealed that Trisk 95 overexpression suppresses depolarization-induced calcium transients on primary cultures of rat skeletal muscle while the caffeine-induced calcium release remains unaffected. To date there are no data on how Trisk 95 overexpression or downregulation would affect the elementary events of calcium release (ECRE). In the present study spontaneous ECRE and calcium transients were studied on C2C12 cells and on primary cultures of skeletal muscle. Liposome- or adenovirus-mediated Trisk 95 overexpression and RNA interference with triadin translation were used to change the level of the protein in these cells. Stable overexpression of Trisk 95 in C2C12 cells significantly decreased the amplitude and frequency of calcium sparks, and the frequency of embers. Similarly, adenoviral transfection of Trisk 95 in primary cultures of mouse skeletal muscle cells significantly decreased both the frequency and the amplitude (0.26 ± 0.01 Hz and 0.56 ± 0.03 DF/F0, n = 71) of spontaneous calcium transients as compared to control myotubes (0.75 ± 0.04 Hz and 0.68 ± 0.02 DF/F0, n = 169). Primary cultures of rat skeletal muscle cells expressing endogenous triadin 95 readily generated spontaneous calcium transients (1.01 ± 0.22 Hz and 0.62 ± 0.05 DF/F0, n = 553), but rarely produced calcium sparks. Transfection of these myotubes with specific shRNA sequence significantly reduced the triadin-specific immunopositivity. Functional experiments on these cells revealed that ECRE appeared with higher frequency. The results suggest that Trisk 95 negatively regulates excitation-contraction coupling by suppressing the elementary calcium release events.