β-carotene hydroxylase (BCH) has been implicated as a key enzyme conferring a stress tolerant mechanism in plants by production of carotenoids, which serve as protectants against photoinhibition and precursor of ABA biosynthesis. We previously cloned a gene encoding a novel cytosolic form of BCH (GmBCH1) from soybean (Glycine max) whose expression increased during nodulation with Bradyrhizobium japonicum. In the present work we extended our study to three GmBCHs as soybean is an allotetraploid, and examined their possible role(s) in nodule development. In situ hybridization revealed the expression of three GmBCHs (GmBCH1, GmBCH2, and GmBCH3) in the infected cells of root nodules, and their enzymatic activities were confirmed by functional assays in E. coli. Localization of GmBCHs by transfecting Arabidopsis protoplasts with GFP fusions and by EM immunogold detection in soybean nodules indicated that GmBCH2 and GmBCH3 were present in plastids while GmBCH1 appeared to be cytosolic. RNAi of the GmBCHs severely impaired nitrogen fixation as well as nodule development. Surprisingly, we failed to detect zeaxanthin, a product of GmBCH, or any other carotenoids, in nodules. We therefore examined the possibility that most of the carotenoids in nodules are converted or cleaved to other compounds. We detected the expression of some carotenoid cleavage dioxygenases (GmCCDs) in wild-type nodules, and also a reduced amount of zeaxanthin in GmCCD8-expressing E. coli, suggesting cleavage of the carotenoid. In view of these findings we propose that carotenoids such as zeaxanthin synthesized in root nodules are cleaved by GmCCDs, and we discuss the possible roles of the carotenoid cleavage products in nodulation.