Objective: To investigate the effects of Hedyotis diffusa Willd (HDW) on the proliferation, cell cycle, apoptosis, migration and invasion of human renal carcinoma cells, and to explore the possible mechanisms. Methods: After human renal adenocarcinoma ACHN cells and human renal proximal tubule HK-2 cells were treated with HDW for 24 h, the cell proliferation was detected by CCK-8 assay, and the cell cycle and apoptosis were detected by FCM assay. The phenotypes of ACHN and HK-2 cells treated with HDW were observed by light microscopy and rhodamine staining. The expressions of mesenchymal-epithelial transition (MET)-related proteins were detected by immunofluorescence staining and Western blotting, respectively. The effects of HDW on the migration and invasion of ACHN and HK-2 cells were detected by wound-healing and Transwell chamber assay. RNAsequencing was used to detect the effect of HDW on the transcriptome in ACHN cells. The expression levels of RAP1 pathway-related genes [RAP1 GTPase activating protein (RAP1GAP), RAS guanyl releasing protein 2 (RASGRP2), RAP guanine nucleotide exchange factor 3 (RAPGEF3), membrane-associated guanylate kinase, WW and PDZ domain containing 1 (MAGI1) and G protein subunit alpha i1 (GNAI1)] were detected by real-time fluorescent quantitative PCR. The expression levels of mitogen-activate protein kinase (MAPK) pathway-related proteins [c-Jun N -terminal kinase (JNK), phospho-JNK (p-JNK), protein kinase B (PKB, Akt), phospho-Akt (p-Akt), extracellular signalregulated kinase (ERK) and phospho-ERK (p-ERK)] were detected by Western blotting. Results: HDW selectively inhibited the proliferation of ACHN cells (P < 0.01), blocked cell cycle at S-phase (P < 0.01), and induced apoptosis (P < 0.01). After treatment with HDW, the morphology of ACHN cells significantly changed. HDW promoted the expressions of MET-related proteins (E-cadherin 1 and β-catenin) in ACHN cells, and inhibited the expressions of epithelial-mesenchymal transition (EMT)-related proteins (Vimentin and Snail1) (all P < 0.01). HDW inhibited the migration and invasion of ACHN and HK-2 cells (all P < 0.01), and there was no significant difference between ACHN and HK-2 cells. HDW affected the expressions of cell cycle-related genes and transcription factor E2F and Myc target genes, and activated the p53 signaling pathway. HDW significantly downregulated the expression levels of RAP1GAP, RASGRP2, RAPGEF3, MAGI1 and GNAI1 (all P < 0.000 1) and the phosphorylation level of JNK in ACHN cells. Conclusion: HDW may selectively inhibit cell proliferation, and promote MET, cell cycle arrest and apoptosis of ACHN cells by inhibiting RAP1-JNK signaling pathway.