A systematic investigation in isolated 5-hydroxyisoxazole–water complexes (5-HIO·(H 2 O) n n=1–3) is performed at the DFT level, employing B3LYP/6-31G(d, p) basis set. Single-point energy calculations are also performed at the MP2 level using B3LYP/6-31G(d, p) optimized geometries and the 6-311++G(d, p) basis set. The computational results show that the keto tautomer K 2 is the most stable isomer in the gas phase, and the tautomer K 1 to be the next most stable tautomer. Hydrogen bonding between HIO and the water molecule(s) will dramatically lower the barrier by a concerted multiple proton transfer mechanism. The proton transfer process of 3WE cis ↔3WK 1 and 2WE trans ↔2WK 2 is found to be more efficient in two tautomerization, and the barrier heights are 7.03 and 14.15kcal/mol at B3LYP/6-31G(d, p) level, respectively. However, the proton transfer reaction between E cis and K 1 cannot happen without solvent-assisted.