As a result of screening for 20S proteasome inhibitors, novel cyclic peptides TMC-95A and its diastereomers TMC-95B to D were isolated from the fermentation broth of Apiospora montagnei Sacc. TC1093. This study describes the taxonomy of the producing strain, production, isolation, and biological activities of TMC-95s. TMC-95A inhibits the chymotrypsin-like (ChT-L), trypsin-like (T-L), and peptidylglutamyl-peptide hydrolyzing (PGPH) activities of 20S proteasome with IC50 values of 5.4nM, 200nM, and 60nM, respectively. TMC-95B exhibits similar inhibitory activity to TMC-95A, while TMC-95C and D are 20 to 150 times weaker. TMC-95A does not inhibit m-calpain, cathepsin L, and trypsin at 30 μM, indicating high selectivity for proteasome. Double reciprocal Lineweaver-Burk plot analysis shows TMC-95A acts as a competitive inhibitor against ChT-L activity with a Ki value of 2.3nM. The stereochemistry at C-7 is essential for inhibitory activity, as TMC-95C and D (differing in C-7 stereochemistry from A and B) have significantly weaker activity. TMC-95A is a novel, specific proteasome inhibitor with a unique structure, serving as a valuable tool for proteasome research. Given the role of 20S proteasome in NF-κB activation, MHC class I ligand processing, and protein breakdown in pathological muscle wasting, TMC-95A and B are expected to have therapeutic potential for inflammatory, autoimmune diseases, and conditions like cancer cachexia, diabetes, and sepsis.