Vibrio parahaemolyticus is an important human pathogen that is currently the leading cause of shellfish-borne gastroenteritis in the world. Particularly, the pandemic strain has the capacity to induce cytotoxicity and enterotoxicity through its Type 3 Secretion System (T3SS2) that leads to massive cell death. However, the specific mechanism by which the T3SS2 induces cell death remains unclear and its contribution to mitochondrial stress is not fully understood. In this work, we evaluated the contribution of the T3SS2 of V. parahaemolyticus in generating mitochondrial stress during infection in human intestinal HT-29 cells. To evaluate the contribution of the T3SS2 of V. parahaemolyticus in mitochondrial stress, infection assays were carried out to evaluate mitochondrial transition pore opening, mitochondrial fragmentation, ATP quantification, and cell viability during infection. Our results showed that the Delta vscn1 (T3SS2+) mutant strain contributes to generating the sustained opening of the mitochondrial transition pore. Furthermore, it generates perturbations in the ATP production in infected cells, leading to a significant decrease in cell viability and loss of membrane integrity. Our results suggest that the T3SS2 from V. parahaemolyticus plays a role in generating mitochondrial stress that leads to cell death in human intestinal HT-29 cells. It is important to highlight that this study represents the first report indicating the possible role of the V. parahaemolyticus T3SS2 and its effector proteins involvement in generating mitochondrial stress, its impact on the mitochondrial pore, and its effect on ATP production in human cells.