Histone deacetylase 11 (HDAC11) is the most recently identified member of HDAC family that currently attracts the interest as a target in medicinal treatments of cancer and immune-related disorders. Moreover, potential applications of HDAC11 inhibition in the food industry are intensively studied. However, current understanding of its physiological role, enzymatic activity and 3D structure remains limited. This lack of knowledge impedes progress in the development of highly specific reagents for targeting the enzyme. To gain a closer look at HDAC11 function and structure, our study expanded to include HDAC11 orthologs. Specific sequence motifs were engaged to search for and identify HDAC11 variants across the entire spectrum of life. Phylogenetic analysis revealed the existence of two distinct isoforms of HDAC11. We selected number of HDAC11 variants from various phylogenetic clades to create a representative set for further biochemical characterization, thus shedding light on the evolution of HDAC11 substrate specificity. The data suggested significant diversity of substrate specificity between identified HDAC11 isoforms. Furthermore, X-ray analysis of an ancestral ortholog provided detailed insights into structural features of HDAC11. This data holds promise for enhancing our understanding of HDAC11 function and facilitating the development of inhibitors tailored to specific isoforms and organisms.