Pituitary prolactinoma is one of the most complicated and fatally pathogenic pituitary adenomas. Therefore, there is an urgent need to improve our understanding of the underlying molecular mechanism that drives the initiation, progression, and metastasis of pituitary prolactinoma. The aim of the present study was to identify the key genes and signaling pathways associated with pituitary prolactinoma using bioinformatics analysis. Transcriptome microarray dataset GSE119063 was downloaded from Gene Expression Omnibus (GEO) database. Limma package in R software was used to screen DEGs. Pathway and Gene ontology (GO) enrichment analysis were conducted to identify the biological role of DEGs. A protein-protein interaction (PPI) network was constructed and analyzed by using HIPPIE database and Cytoscape software. Module analyses was performed. In addition, a target gene-miRNA regulatory network and target gene-TF regulatory network were constructed by using NetworkAnalyst and Cytoscape software. Finally, validation of hub genes by receiver operating characteristic (ROC) curve analysis. A total of 989 DEGs were identified, including 461 up regulated genes and 528 down regulated genes. Pathway enrichment analysis showed that the DEGs were significantly enriched in the retinoate biosynthesis II, signaling pathways regulating pluripotency of stem cells, ALK2 signaling events, vitamin D3 biosynthesis, cell cycle and aurora B signaling. Gene Ontology (GO) enrichment analysis showed that the DEGs were significantly enriched in the sensory organ morphogenesis, extracellular matrix, hormone activity, nuclear division, condensed chromosome and microtubule binding. In the PPI network and modules, SOX2, PRSS45, CLTC, PLK1, B4GALT6, RUNX1 and GTSE1 were considered as hub genes. In the target gene-miRNA regulatory network and target gene-TF regulatory network, LINC00598, SOX4, IRX1 and UNC13A were considered as hub genes. Using integrated bioinformatics analysis, we identified candidate genes in pituitary prolactinoma, which might improve our understanding of the molecular mechanisms of pituitary prolactinoma.