The isoquinoline alkaloid chelerythrine is described as an inhibitor of SERCA. The ATPase inhibition presented two non-competitive components, Ki1 = 1, 2 μM and Ki2 = 26 μM. Conversely, chelerythrine presented a dual effect on the p-nitrophenylphosphatase (pNPPase) of SERCA. Ca2+-dependent pNPPase was activated up to ∼5 μM chelerythrine with inhibition thereafter. Ca2+-independent pNPPase was solely inhibited. The phosphorylation of SERCA with ATP reached half-inhibition with 10 μM chelerythrine and did not parallel the decrease of ATPase activity. In contrast, chelerythrine up to 50 μM increased the phosphorylation by Pi. Cross-linking of SERCA with glutaraldehyde was counteracted by high concentrations of chelerythrine. The controlled tryptic digestion of SERCA shows that the low-affinity binding of chelerythrine evoked an E2-like pattern. Our data indicate a non-competitive inhibition of ATP hydrolysis that favors buildup of the E2-conformers of the enzyme. Chelerythrine as low as 0.5–1.5 μM resulted in an increase of intracellular Ca2+ on cultured PBMC cells. The inhibition of SERCA and the loss of cell Ca2+ homeostasis could in part be responsible for some described cytotoxic effects of the alkaloid. Thus, the choice of chelerythrine as a PKC-inhibitor should consider its potential cytotoxicity due to the alkaloid’s effects on SERCA.