SET　and　MYND　domain-containing　protein　2　(SMYD2),　an　identified　protein-lysine　methyltransferase,　is　key　for　bladder　cancer　(BC)　progression.　The　tumor-formation　capacity　and　metastatic　potential　of　bladder　cancer　stem　cells　(BCSCs)　are　due　to　their　stemness　characteristics.　The　present　study　explores　the　mechanism　of　SMYD2　in　promoting　BCSC　stemness　maintenance　by　pyrroline-5-carboxylate　reductase　1　(PYCR1).　BC　cells　were　treated　with　PYCR1,　SMYD2　and　putative　kinase　1　(PINK1)　small　interfering　(si)RNA,　pcDNA3.1-PYCR1　and　pcDNA3.1-SMYD2.　Mito-Tracker　Green　and　light　chain-3B　(LC3B)　expression,　in　vitro　colony　formation　ability　and　tumor　stemness　were　assessed,　as　well　as　histone　H3　lysine　4　trimethylation　(H3K4me3)　enrichment　and　PYCR1,　SMYD2,　H3K4me3,　LC3B　II/I,　p62,　PINK1,　Parkin,　Nanog　and　SRY-box　transcription　factor　2　(Sox2)　expression.　A　nude　mouse　xenograft　model　was　used　for　in　vivo　verification.　PYCR1　mRNA　and　protein　expression　were　elevated　in　BCSCs.　Following　PYCR1　or　SMYD2　siRNA　treatment,　PYCR1,　SMYD2　and　CD44+CD33+　expression,　cancer　cell　colony　formation,　number　of　tumor　spheres　and　Nanog　and　Sox2　expression　were　decreased,　but　pcDNA3.1-PYCR1　or　pcDNA3.1-SMYD2　transfection　enhanced　BCSC　stemness　maintenance.　SMYD2　was　associated　with　PYCR1　expression.　SMYD2　upregulated　PYCR1　expression　through　H3K4me3,　subsequently　activating　the　PINK1/Parkin　mitophagy　pathway,　which　supports　maintenance　of　BCSC　stemness.