Mitochondrial diseases are clinically heterogeneous which involve multiple systems such as organs that are highly dependent on metabolism. Dysfunction of mtDNA is the main cause of mitochondrial diseases that trigger inflammation and immune responses. Here, we aim to identify the biological function and pathways of MEFs with the dysfunction of mtDNA through deletion of YME1L. The gene expression profiles of GSE161735 dataset were originally created by the Illumina NovaSeq 6000 (Mus musculus) for gene biogenesis and function panel. The biological and functional pathways were analyzed by the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG), Gene Ontology (GO), and Reactom visual map. KEGG and GO results showed the metabolism and immune responses were mostly affected by the loss of mtDNA. Moreover, we discovered several interacting genes including POLR2F, HIST1H2BJ, PPP1CC, HOXB4, ARG1, APITD1, BUB1B, POLR2K, HOXC4, and HOXB3 were involved in the regulation of metabolic or cancer diseases. Further, we predicted several regulators that had the ability to affect mitochondria during the dysfunction of mtDNA by L1000fwd analysis. Thus, this study provides further insights into the mechanism of mtDNA in metabolic diseases.