The herpes simplex virus 1 (HSV-1) UL7 gene is highly conserved among herpesviridae. Since the construction of recombinant HSV-1 with a mutation in the UL7 gene has not been reported, the involvement of HSV-1 UL7 in viral replication has been unclear. In this study, we succeeded in generating a UL7 null HSV-1 mutant virus, MT102, and characterized it. Our results were as follows. (i) In Vero cells, MT102 was replication-competent, but formed smaller plaques and yielded 10- to 100-fold fewer progeny than the wild-type virus, depending on the multiplicity of infection. (ii) Using mass spectrometry-based proteomics technology, we identified a cellular mitochondrial protein, adenine nucleotide translocator 2 (ANT2), as a UL7-interacting partner. (iii) When ANT2 was transiently expressed in COS-7 cells infected with HSV-1, ANT2 was specifically co-precipitated with UL7. (iv) Cell fractionation experiments with HSV-1-infected cells detected the UL7 protein in both the mitochondrial and cytosolic fractions, whereas ANT2 was detected only in the mitochondrial fraction. These results indicate the importance of HSV-1 UL7's involvement in viral replication and demonstrate that it interacts with ANT2 in infected cells. The potential biological significance of the interaction between UL7 and ANT2 is discussed.