Uracil phosphoribosyltransferase (UPRT) enzyme has immense potential in prodrug-mediated cancer therapy. Molecular docking and enzyme inhibition studies are important for understanding drug–protein interaction in modern drug design. Herein, we experimentally determined the enzyme inhibition constant (Ki) of 5-fluorouracil (5FU)—a competitive inhibitor of uracil, on UPRT purified from Escherichia coli. In silico experiments were performed on X-ray crystallography structure of UPRT to establish ligand protein interactions, where uracil and its selective inhibitor 5FU were docked computationally to the active site of UPRT enzyme. DOCK 5.2 was employed to dock the ligands to 1,250 molecular dynamics (MDs) snapshots of UPRT. The results highlighted the key residues of UPRT involved in the binding with the ligands. The findings are important for rational design of mutant E. coli UPRT with high selectivity towards the prodrug for suicide gene therapy.