GOLPH3 is a peripheral membrane protein that is significantly associated with the development of cancer. However, the signaling pathways and mechanisms of GOLPH3 mediated cancer are still unclear. Here, our objective is to identify the essential molecules and signaling pathways by analyzing the RNA-seq data. The GSE196875 was produced by the Illumina NovaSeq 6000 (Homo sapiens). The KEGG and GO analyses indicated that axon guidance, signaling pathways regulating pluripotency of stem cells, and circadian entrainment are the major affected processes during the development of prostate cancer with GOLPH3 overexpression. Moreover, the ten interacting molecules were figured out including BMP2, RAC2, CACNA1C, PAX6, COL4A1, SPARC, GRIN1, CAV3, COL5A1, COL4A2. Thus, our study provides novel knowledge on the GOLPH3 affected cancer cells.