Updated publication reference for PubMed record(s): 26750096. The significance of non-histone lysine methylation in cell biology and human disease is an emerging area of research exploration, and small molecule inhibitors that selectively and potently target “writers” and “erasers” of methyl-lysine, such as the protein lysine mono-methyltransferase SMYD2, are active areas of drug discovery. It is therefore essential to clarify the substrates of these enzymes in cellular contexts in order to advance and to correctly understand the cellular mechanism(s) of action of these targets. Here, using stable isotopic labelling of amino acids in cell culture (SILAC) coupled with immunoaffinity enrichment of mono-methyl-lysine (Kme1) peptides and mass spectrometry, we report the most comprehensive and large-scale proteomic study of protein mono-methylation, comprising a total of 1032 Kme1 sites identified in esophageal squamous cell carcinoma (ESCC) cells and 1861 Kme1 sites in ESCC cells overexpressing SMYD2. Among these sites was a subset of 35 robust Kme1 sites that were potently down-regulated by both shRNA-mediated knockdown of SMYD2 and LLY-507, a small molecule inhibitor of SMYD2. In addition, we report specific protein sequence motifs that were enriched in Kme1 sites and that were also directly regulated by endogenous SMYD2 activity, indicating that the diversity of SMYD2 substrate specificity of SMYD2 exceeds previous expectations. Furthermore, we reveal that BTF3 and PDAP1 are novel and direct substrates of SMYD2, as well as AHNAK and AHNAK2, two large and highly-repetitive proteins directly and extensively mono-methylated by SMYD2 at several lysine residues. Collectively, our findings provide novel quantitative and insights into the cellular activity and substrate recognition of SMYD2 as well as the global landscape and regulation of protein mono-methylation in cells.