Fibrosis, characterized by the abnormal deposition of extracellular matrix, can cause organ dysfunction and mortality. However, research on the mechanisms of fibrosis, especially that focusing on the identification of drug targets, is currently still lacking. This study is the first to reveal that a small molecule (YTB-8) derived from the natural β-carboline alkaloid flazin has potent protective effects against renal fibrosis in vitro and in vivo. The cellular target of YTB-8, a nuclear and endoplasmic reticulum membrane protein known as transmembrane protein 43 (TMEM43), was identified by activity-based protein profiling (ABPP) strategy in a renal fibrosis model. YTB-8 was found to interact with amino acids Arg312 and Leu382 of TMEM43 and exert anti-renal fibrosis effects by suppressing the NF-κB and TGF-β/Smad signaling pathways. Utilization of simulated TMEM43 and its pocket structure for virtual screening, followed by in vitro and in vivo detection, led to us identifying two FDA-approved drugs, drospirenone and adapalene, with anti-renal fibrosis efficacy. TMEM43 was also found to be the target of YTB-8 in its alleviation of lung, liver, and skin fibrosis in vitro, verifying the value of TMEM43 as a general target for new-generation drug design relating to organ fibrosis. Collectively, these findings revealed TMEM43 to be a promising universal cellular target for organ fibrosis intervention.