Abstract: Lysine methylation of histones and non-histones plays a pivotal role in diverse cellular processes. The SMYD (SET and MYND domain) family methyltransferases can methylate various histone and non-histone substrates in mammalian systems, implicated in HSP90 methylation, myofilament organization, cancer inhibition, and gene transcription regulation. To resolve controversies concerning SMYD's substrates and functions, we studied SMYD1 (TTHERM_00578660), the only homologue of SMYD in the unicellular eukaryote Tetrahymena thermophila. We epitope-tagged SMYD1, and analyzed its localization and interactome. We also characterized ΔSMYD1 cells, focusing on the replication and transcription phenotype. Our results show that: (1) SMYD1 is present in both cytoplasm and transcriptionally active macronucleus and shuttles between cytoplasm and macronucleus, suggesting its potential association with both histone and non-histone substrates; (2) SMYD1 is involved in DNA replication and regulates transcription of metabolism-related genes; (3) HSP90 is a potential substrate for SMYD1 and it may regulate target selection of HSP90, leading to pleiotropic effects in both the cytoplasm and the nucleus.