Background: Long-term exposure to ultraviolet (UV) radiation can cause cutaneous squamous cell carcinoma (cSCC), which is one of the most common malignant cancers worldwide. Actinic keratosis (AK) is generally considered a precancerous lesion of cSCC. However, the pathogenesis and oncogenic processes of AK and cSCC remain elusive, especially in the context of photodamage. Methods: In this study, transcriptome sequencing was performed on AK, cSCC, normal sun-exposed skin (NES) tissues, and normal non-sun-exposed skin (NNS) from 24 individuals. Bioinformatics analysis to identify the differentially expressed genes (DEGs) of 4 groups, and potential key genes of cSCC were validated by real-time quantitative reverse transcription PCR (qRT-PCR). Results: A total of 46,930 genes were differentially expressed in the 4 groups, including 127 genes that were differentially expressed between NES and NNS, 420 DEGs in AK compared to NES, 1,658 DEGs in cSCC compared to NES, and 1,389 DEGs in cSCC compared to AK. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis suggested that the DEGs are involved in multiple pathways, including extracellular matrix (ECM)-receptor interaction, immune, inflammatory, microbial infection, and other related pathways. Finally, 5 new genes (HEPHL1, FBN2, SULF1, SULF2, and TCN1) were confirmed significantly upregulated in cSCC. Conclusions: Using transcriptome sequencing and integrated bioinformatical analysis, we have identified key DEGs and pathways in cSCC, which could improve our understanding of the cause and underlying molecular events of AK and cSCC. HEPHL1, FBN2, SULF1, SULF2, and TCN1 may be novel potential biomarkers and therapeutic targets of cSCC.