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Two major bile acids were isolated from the gallbladder bile of two hornbill species from the Bucerotidae family of the avian order Bucerotiformes Buceros bicornis (great hornbill) and Penelopides panini (Visayan tarictic hornbill). Their structures were determined to be 3α,7α,24‐dihydroxy‐5β‐cholestan‐27‐oic acid and its 12α‐hydroxy derivative, 3α,7α,12α,24‐tetrahydroxy‐5β‐cholestan‐27‐oic acid (varanic acid, VA), both present in bile as their corresponding taurine amidates. The four diastereomers of varanic acid were synthesized and their assigned structures were confirmed by X‐ray crystallographic analysis. VA and its 12‐deoxy derivative were found to have a (24R,25S)‐configuration. 13 additional hornbill species were also analyzed by HPLC and showed similar bile acid patterns to B. bicornis and P. panini. The previous stereochemical assignment for (24R,25S)‐VA isolated from the bile of varanid lizards and the Gila monster should now be revised to the (24S,25S)‐configuration.