DHX32 has an overall similarity to the DHX family of RNA helicases but with a novel helicase domain and nuclear and mitochondrial localizations. The expression of DHX32 is highly regulated during lymphocyte activation and is dysregulated in lymphoid malignancies. In this study, we report our finding of an altered subcellular localization of heat shock protein 60 (Hsp60) in Jurkat-DHX32 cell line in which DHX32 is constitutively expressed. Two-dimensional gel electrophoresis followed by mass spectrometry, electron microscopic immunocytochemistry, and immunoblot analysis showed mainly cytoplasmic localization of Hsp60 in Jurkat-DHX32 cells instead of its mainly mitochondrial localization in control cells. No significant changes were detected in the mitochondrial ultra-structure and the mitochondrial membrane potential activity as a result of dysregulated DHX32 expression. The subcellular distribution of several mitochondrial proteins including cytochrome c, peroxiredoxin 3, manganese superoxide dismutase, Leucine-Rich PentatricoPeptide Repeat Cassette and the 49-kDa subunit of the mitochondrial respiratory Complex I were similar in control and Jurkat-DHX32 cells ruling out non-specific cytoplasmic leakage of mitochondrial proteins. No significant changes in the expression of Hsp60 transcript or total cellular protein were detected. These findings suggest that dysregulated expression of DHX32 might lead to as of yet unknown changes in mitochondrial homeostasis manifested by cytoplasmic redistribution of the molecular chaperon Hsp60.