Fusarium nematophilum NQ8GII4 is an endophytic fungus isolated from the root of healthy wolfberry (Lycium barbarum). Previous studies have reported that NQ8GII4 could dwell in wolfberry roots and enhance the defense responses in wolfberry against root rot, which is caused by F. oxysporum. To further elucidate the molecular mechanism of wolfberry disease resistance induced by NQ8GII4, in the present study, we adopted RNA sequencing analysis to profile the transcriptome of wolfberry response to NQ8GII4 infestation over a time course of 3 and 7 days post-inoculation (dpi). Gene ontology (GO) enrichment analysis revealed that DEGs were enriched related to biological regulation, response to stimulus, signaling, detoxification, immune system process, transporter activity, electron carrier activity, transcription factor activity, nucleic acid binding transcription factor, and antioxidant activity. Through Kyoto encyclopedia of genes and genomes (KEGG) analysis, it was found that many of these DEGs were enriched in pathways related to plant-pathogen interactions, hormone signal transduction, and phenylpropanoid biosynthesis pathway in wolfberry. This suggests that innate immunity, phytohormone signaling, and numerous phenylpropanoid compounds, which comprise a complex defense network in wolfberry. Chloroplast 50S ribosomal proteins (50S RP) were consistently located at the core position of the response in wolfberry following infestation with NQ8GII4 analyzed by protein-protein interaction (PPI) network. This study elucidated the molecular mechanism underlying the interaction between NQ8GII4 and wolfberry, clarified the wolfberry immune response network to endophytic fungi infestation, identified candidate resistance genes in wolfberry, and provided a fundamental date for subsequent work.