Acetohydroxyacid synthase (AHAS) as the first common enzyme in the brunched-chain amino acids biosynthesis is the target of several classes of commercial herbicide. AHAS is normally composed of a larger catalytic subunit with FAD (flavin adenine dinucleotide), ThDP (thiamine diphosphate) and Mg²⁺ as cofactors and a smaller regulatory subunit bound to the end-product feedback signals such as valine, leucine or isoleucine to down regulate the holoenzyme activities. The Escherichia coli ilvN gene that encodes the regulatory subunit of AHAS Ⅰ was cloned into pET28a and expressed in soluble form at high levels in E. coli strain BL21 (DE3). The protein was purified using Ni²⁺-chelating chromatography followed by size-exclusion chromatography. Crystals of IlvN protein were obtained and diffracted to 2.6 Å. To further study the regulatory mechanism, crystals of IlvN co-crystallized with its effector valine were also obtained and diffracted to 3.0 Å.