Auxin regulates cell division and elongation of the primordial cells through its concentration and then shaped the plant architecture. Cell division and elongation form the intode of soybean and result in different plant heights and lodging resistance. Yet the mechanisms behind are unclear in soybean. To elucidate the mechanism of the concentration difference of auxin related to stem development in soybean, samples of apical shoot, elongation zone, and mature zone from the developing stems of soybean seedlings, Charleston, were harvested and measured for auxin concentration distributions and metabolites to identify the common underlying mechanisms responsible for concentration difference of auxin. Distribution of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and methylindole-3-acetic acid (Me-IAA) were determined and auxin concentration distributions were found to have a complex regulation mechanism. The concentrations of IAA and Me-IAA in apical shoot were significantly different between elongation zone and mature zone resulting in an IAA gradient. Tryptophan dependent pathway from tryptamine directly to IAA or through indole-3-acetonitrile to IAA and from indole-3-propionic acid (IPA) to IAA were three primary IAA synthesis pathways. Moreover, some plant metabolites from flavonoid and phenylpropanoid synthesis pathways showed similar or reverse gradient and should involve in auxin homeostasis and concentration difference. All the data give the first insight in the concentration difference and homeostasis of auxin in soybean seedlings and facilitate a deeper understanding of the molecular mechanism of stem development and growth. The gathered information also helps to elucidate how plant height is formed in soybean and what strategy should be adopted to regulate the lodging resistance in soybean.