Chronic glomerulonephritis (CGN) is a disease occurred in glomeruli. The mechanism of CGN is regarded to be involved in a range of inflammatory responses. MicroRNA-339-5p (miR-339-5p) has been reported to be involved in inflammatory responses in many diseases. However, the role of miR-339-5p in CGN remains unclear. The purpose of this study was to investigate the role of miR-339-5p in lipopolysaccharide (LPS)-induced nephritis injury in vitro. The real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) and western blot (WB) were used to detect the expression of miR-339-5p and Syk/Ras/c-Fos pathway. Double luciferase was performed to identify targeted binding of miR-339-5p to Syk. Cell counting kit-8 (CCK-8) and flow cytometry were used to observe cell viability and cell cycle. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the concentrations of inflammatory cytokines IL-1β, IL-10, IL-6, and TNF-α. Lipopolysaccharide (LPS) could increase HBZY-1 (rat mesangial cells) cell viability, decrease the G2 phase, and promote cell proliferation and accelerate inflammatory cytokine. However, overexpression of miR-339-5p could inhibit LPS-induced HBZY-1 cell viability, decrease the expression of Syk/Ras/c-Fos signaling pathway, downregulate the expression level of inflammatory cytokines, increase the G2 phase, and inhibit cell proliferation. miR-339-5p could inhibit the proliferation and inflammation of the rat mesangial cells through regulating Syk/Ras/c-Fos signaling pathway.