Mincle is a C-type lectin receptor of the innate immune system with the ability to sense pathogens and commensals through lipidic metabolites. While a growing number of bacterial glycolipids have been discovered that can signal through human Mincle, no fungal metabolites are known that can signal through the human form of this receptor. We report the total synthesis of a complex β-1,2-mannosyloxymannitol glycolipid from Malassezia pachydermatis 44-2, which was reported to signal through the murine Mincle receptor. Assembly of 44-2 was achieved through a highly convergent route that exploits symmetry elements inherent within this molecule and delineation of conditions that maintain the delicate l-mannitol triester-triol array. We show that 44-2 is a potent agonist of human Mincle signaling and constitutes the first fungal metabolite identified that can signal through the human Mincle receptor, providing new insights into antifungal immunity.