Nicotinic acid adenine dinucleotide phosphate (NAADP) gates two-pore channels 1 and 2 (TPC1 and TPC2) to elicit endo-lysosomal (EL) Ca²⁺ release. NAADP-induced EL Ca²⁺ signals may be amplified by the endoplasmic reticulum (ER) through the Ca²⁺-induced Ca²⁺ release mechanism (CICR). Herein, we aimed at assessing for the first time the role of EL Ca²⁺ signaling in primary cultures of human metastatic colorectal carcinoma (mCRC) by exploiting Ca²⁺ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe β-naphthylamide (GPN), and nigericin, which dissipates the ΔpH which drives Ca²⁺ refilling of acidic organelles, caused massive Ca²⁺ release in the presence of a functional inositol-1,4,5-trisphosphate (InsP₃)-sensitive ER Ca²⁺ store. Liposomal delivery of NAADP induced a transient Ca²⁺ release that was reduced by GPN and NED-19, a selective TPC antagonist. Pharmacological and genetic manipulations revealed that the Ca²⁺ response to NAADP was triggered by TPC1, the most expressed TPC isoform in mCRC cells, and required ER-embedded InsP₃ receptors. Finally, NED-19 and genetic silencing of TPC1 reduced fetal calf serum-induced Ca²⁺ signals, proliferation, and extracellular signal-regulated kinase and Akt phoshorylation in mCRC cells. These data demonstrate that NAADP-gated TPC1 could be regarded as a novel target for alternative therapies to treat mCRC.