AbstractIn recent years, carbohydrate epimerases have attracted increasing attention as promising biocatalysts for the production of specialty sugars and derivatives. The vast majority of these enzymes are active on nucleotide‐activated sugars, rather than on their free counterparts. Although such epimerases are known to have a clear preference for a particular nucleotide (UDP, GDP, CDP, or ADP), very little is known about the determinants of the respective specificities. In this work, sequence motifs are identified that correlate with the different nucleotide specificities in one of the main epimerase superfamilies, carbohydrate epimerase 1 (CEP1). To confirm their relevance, GDP‐ and CDP‐specific residues are introduced into the UDP‐glucose 4‐epimerase from Thermus thermophilus, resulting in a 3‐fold and 13‐fold reduction in KM for GDP‐Glc and CDP‐Glc, respectively. Moreover, several variants are severely crippled in UDP‐Glc activity, which further underlines the crucial role of the identified positions. Hence, the analysis should prove to be valuable for the further exploration and application of epimerases involved in carbohydrate synthesis.