Lysosomes and lysosomal Ca2+ play crucial roles in cellular homeostasis and drug resistance. The lysosomal Ca2+ channel transient receptor potential mucolipin 3 (TRPML3; also known as mucolipin‑3 or MCOLN3) is a key regulator of autophagy and membrane trafficking; however, its role in tyrosine kinase inhibitor (TKI) resistance remains unclear. The contribution of TRPML3 to osimertinib resistance in non‑small cell lung cancer (NSCLC) was therefore assessed. Using publicly available RNA sequencing data, including profiles from clinical samples before and after osimertinib treatment, TRPML3 expression was measured in lung adenocarcinoma (LUAD) tissues. Additionally, two‑dimensional cell culture of, and three‑dimensional spheroids derived from, NSCLC cell lines were used to elucidate roles of TRPML3 in drug resistance. TRPML3 expression was significantly upregulated in both LUAD tissues from patients with residual disease after osimertinib treatment, as well as in osimertinib‑resistant NSCLC cells. TRPML3 knockdown in resistant PC9 cells restored sensitivity to osimertinib and multiple TKIs; this was replicated in spheroid models. Mechanistically, osimertinib induced intracellular Ca2+ oscillations in PC9 cells via lysosomal Ca2+ release through TRPML3 rather than through TRPML1. In summary, the present findings suggest that elevated TRPML3 expression compensates for TRPML1 to maintain lysosomal acidity and biogenesis during TKI treatment, facilitating drug sequestration and resistance and identifying TRPML3 as a potential target for overcoming osimertinib resistance in NSCLC.