Based on the antioxidant properties of Phellinus igniarius (DC. Ex Fr.) Quel (P. igniarius), this study aims to investigate the protective effect and mechanism of total flavonoids of Phellinus igniarius (TFPI) on the oxidative damage of HK-2 cells induced by monosodium urate (MSU). The GO and KEGG enrichment analyses predicted the potential targets and pathways of TFPI in the treatment of hyperuricaemia (HUA). We used MSU to stimulate HK-2 cells to establish a HUA model. Cell viability, lactate dehydrogenase (LDH) assay, and reactive oxygen species (ROS) assay were performed. Cell nuclear morphology was detected with DAPI and Annexin V-FITC. The activity of superoxide dismutase (SOD) and malondialdehyde (MDA) was analyzed. The expression of Keap 1, Nrf2, HO-1, Bip, PERK, ATF4, CHOP, BAX, Bcl-2, and cleaved caspase 3 was performed with western blot. The results of network pharmacology showed that the potential targets and pathways were mainly associated with stimulus response, regulation of reactive oxygen species metabolic processes, response to oxidative stress, and regulation of the response to the endoplasmic reticulum (ER) stress pathway. The cell experiment proved that the survival rate of HK-2 cells was dramatically increased after treatment with TFPI. TFPI increased the activity of SOD and decreased the content of MDA and LDH, while scavenging ROS. TFPI increased the transfer of Nrf2 protein from the cytoplasm to the nucleus and decreased the expression level of Bip, PERK, ATF4, CHOP, BAX, and cleaved caspase 3 to attenuate apoptotic cells induced by MSU. TFPI has a good protective effect on MSU-induced oxidative damage in HK-2 cells and can reduce cell apoptosis by regulating ROS-mediated ER stress.