Neutrophils are important in innate immunity and in the initiation of an acute response to infection. Under normal conditions, the mitochondrial membrane potential of neutrophils is low, and neutrophils energy depends fundamentally on glycolysis. In contrast, neutrophils require energy supplied from mitochondrial oxidative phosphorylation (OXPHOS) during infection. The inhibition of mitochondrial OXPHOS blocks the chemotaxis of neutrophils. Here, we examined the mitochondrial morphology of neutrophil-like differentiated HL-60 cells after chemoattractant N-formyl-Met-Leu-Phe (fMLP) stimulation. We found that mitochondrial morphology changes to a tubular form after fMLP stimulation. Mitochondrial OXPHOS activity and mitochondrial complex II significantly increased after fMLP stimulation. On the other hand, the silencing of mitochondrial fusion protein mitofusin 2 (MFN2) suppresses mitochondrial morphological changes. MFN2 silencing suppressed OXPHOS activation and chemotaxis after fMLP stimulation. Furthermore, the silencing of MFN2 associated protein suppresses also mitochondrial morphological changes and chemotaxis upon fMLP stimulation. These results suggest that MFN2 and MFN2 associated protein are involved in chemotaxis of differentiated HL-60 cells.