The preservation of mitochondrial homeostasis plays a critical role in preventing the progression of pathological cardiac hypertrophy to heart failure (HF). Although transient receptor potential mucolipin 1 (TRPML1) has been recently linked to lysosomal homeostasis, its role in pressure overload-induced pathological cardiac hypertrophy remains unclear. Transcriptomic analyses of both mouse and human HF samples revealed the significant downregulation of TRPML1 expression. Cardiomyocyte-specific overexpression and pharmacological activation of TRPML1 markedly improved cardiac function, reduced mitochondrial oxidative stress, and increased energy production. In contrast, cardiomyocyte-specific deletion or pharmacological inhibition of TRPML1 exacerbated cardiac hypertrophy and mitochondrial dysfunction. Further investigations revealed that signal transducer and activator of transcription 5B (Stat5b) is a transcriptional regulator of TRPML1 in the context of cardiac hypertrophy. Mechanistically, the proteomic analysis revealed that the C-terminal domain of TRPML1 directly interacts with the N-terminal domain of voltage-dependent anion channel 1 (VDAC1). This interaction inhibited VDAC1 oligomerization, thereby maintaining mitochondrial calcium (Ca2+) homeostasis and the balance of mitochondrial fusion and fission in hypertrophic cardiomyocytes. The administration of 1,3-bis(4-aminophenyl)urea (NSC 15364) to inhibit VDAC1 oligomerization partially reversed cardiac hypertrophy in TRPML1 knockout mice. These findings highlight TRPML1 as a promising therapeutic target to suppress the progression of pathological cardiac hypertrophy to HF.