UFSP1 (UFM1 specific peptidase 1) is a thiol-dependent cysteine protease that catalyzes two essential functions in UFMylation, a ubiquitin-like post-translational modification pathway 1. UFSP1 mediates both maturation of pro-UFM1 precursors through proteolytic cleavage to expose the C-terminal glycine 2, and deconjugation of UFM1 from target proteins 1. Structurally, UFSP1 contains a papain-like catalytic fold with a conserved Cys active site and an atypical Asp-Pro-His catalytic triad 3. A notable discovery revealed that human UFSP1 is translated from a non-canonical CUG start codon via eIF2A-mediated initiation rather than the annotated AUG, explaining its previous mischaracterization as a pseudogene 1. UFSP1 and UFSP2 exhibit distinct substrate specificities: UFSP1 acts earlier in UFMylation pathway maturation and cleaves potential autoinhibitory modifications on UFC1, while UFSP2 specifically removes UFM1 from ribosomal RPL26 2. Cells lacking both UFSPs show complete loss of UFMylation 2. UFMylation regulates diverse cellular processes including endoplasmic reticulum homeostasis and liver protection in metabolic dysfunction 4. Despite enrichment at neuromuscular junctions, UFSP1 knockout mice show normal development without gross phenotypic abnormalities, suggesting redundancy or context-dependent function 5.