Squamous cell carcinoma arising from mature teratoma (SCC-MT) is a rare ovarian malignancy. The detailed molecular pathology of SCC-MT is not well understood. Moreover, the prognosis of the patients remains poor because no standard treatment has been established. In this study, we performed single-nucleus RNA sequencing and spatial transcriptomics using clinical SCC-MT samples to identify novel therapeutic candidates. snRNA-seq revealed three epithelial cell clusters, of which one was significantly associated with epidermis and keratinocyte development. Moreover, spatial transcriptomics revealed that the epithelial-mesenchymal transition was significantly inhibited, and the MYC and E2F targets were significantly activated in cancer spots on specimen sections. We focused on KLF5, which was one of the upregulated genes in cancer cells, and performed a functional analysis using NOSCC-1, a cell line derived from an SCC-MT. KLF5 downregulation significantly decreased cell proliferation and increased apoptosis. Furthermore, we previously identified miR-145-5p as a downregulated miRNA in SCC-MT. We demonstrated that miR-145-5p overexpression attenuated cell proliferation and decreased KLF5 expression. In conclusion, through multi-omics analyses, we identified unique gene expression profiles of SCC-MT and determined a role for KLF5 in SCC-MT development. Therefore, KLF5-related factors may be novel therapeutic targets, and further studies are needed to improve the diagnosis and treatment of SCC-MT.