3-mercaptopyruvate sulfurtransferase (MPST) is an enzyme implicated in the generation of the gasotransmitter hydrogen sulfide (H2S). Unlike, the other two H2S-synthesizing enzymes cystathionine gamma lyase (CSE) and cystathionine beta synthase (CBS), MPST is found in the mitochondria. However, the mechanisms through which MPST gains access to the mitochondria and its exact localization within this organelle remain unclear. Using immunogold electron microscopy staining, we localized MPST on the inner mitochondrial membrane. To study the pathway of mitochondrial entry for MPST, pharmacological inhibitors of different components of the translocase of outer/inner membrane were used. In line with the observation that ΜPST is found on the inner mitochondrial membrane, inhibition of TIM23 blocked MPST mitochondrial entry. Generation of N-terminally truncated forms of ΜPST did not interfere with the ability of the enzyme to gain access into the mitochondria, suggesting that an N-terminal pre-sequence does not mediate MPST mitochondrial entry. In agreement to this finding, cytosolic and mitochondrial MPST had a similar molecular weight. Interestingly, N-terminally deleted MPST exhibited reduced expression levels, indicating that this part of the enzyme is required for protein stability. Molecular dynamics simulations confirmed that deletion of the first 39 amino acids of the enzyme destabilizes the protein. Our findings reveal that MPST is present on the inner mitochondrial membrane and that its entry into mitochondria does not involve the N-terminus of the protein.