The glycolytic enzyme aldolase is an emerging drug target in diseases such as cancer and protozoan infections which are dependent on a hyperglycolytic phenotype to synthesize adenosine 5'-triphosphate and metabolic precursors for biomass production. To date, structural information for the enzyme in complex with phosphate-derived inhibitors has been lacking. Thus, we determined the crystal structure of mammalian aldolase in complex with naphthalene 2,6-bisphosphate (1) that served as a template for the design of bisphosphonate-based inhibitors, namely, 2-phosphate-naphthalene 6-bisphosphonate (2), 2-naphthol 6-bisphosphonate (3), and 1-phosphate-benzene 4-bisphosphonate (4). All inhibitors targeted the active site, and the most promising lead, 2, exhibited slow-binding inhibition with an overall inhibition constant of ∼38 nM. Compound 2 inhibited proliferation of HeLa cancer cells, whereas HEK293 cells expressing a normal phenotype were not inhibited. The crystal structures delineated the essential features of high-affinity phosphate-derived inhibitors and provide a template for the development of inhibitors with prophylaxis potential.