Renal fibrosis is the final pathway for chronic kidney disease to end-stage renal failure. Non-coding RNAs have been reported to play a crucial role in renal fibrosis. Here, the effects of lncRNA NEAT1 and miR-31 on renal fibrosis and their regulatory mechanism were evaluated. RT-qPCR was used to assess NEAT1, miR-31 and RhoA levels. Western blot was performed to analyze the expression of fibrosis markers, RhoA, ROCK1 and CTGF. RIP, FISH and luciferase reporter assays verified the interaction between miR-31 and NEAT1 or RhoA. Renal fibrosis and injury were observed by Masson and HE staining. The expression level of inflammatory cytokines was detected by ELISA. Immunohistochemistry was performed to examine the expression levels of α-SMA and RhoA in renal tissues. We showed NEAT1 was highly expressed, whereas miR-31 was decreased in renal fibrosis. NEAT1 was found to directly bind miR-31 to positively regulate RhoA expression. Furthermore, NEAT1 silencing inhibited renal fibrosis and inflammation, and suppressed the RhoA/ROCK1 signaling pathway. However, knockdown of miR-31 could reverse these effects. NEAT1 silencing or overexpression of miR-31 alleviated renal fibrosis in vivo. In conclusion, NEAT1 accelerates renal fibrosis progression via negative regulation of miR-31 and the activation of RhoA/ROCK1 pathway, thereby upregulating the expression level of CTGF, providing a theoretical basis for treatment and prognostic evaluation of renal fibrosis.