Undissolved humic acid (HA) has a great retention effect on the migration of hexavalent chromium [Cr(VI)] in soil, and HA functional groups play a predominant role in this process. However, the coupled mode between Cr(VI) retention and HA functional groups reaction is still unclear. In this study, it was found that a fair amount of Cr on HA existed in the forms of ion exchangeable and binding Cr(VI) during the reaction resulting from the ion exchange adsorption and complexation of Cr(VI). According to the results of two-dimensional correlation spectroscopic analysis (2DCOS), HA functional groups participated in the reaction with Cr(VI) in the order of carboxyl ≈ chelated carboxyl > phenol > polysaccharide > methyl, and all the functional groups were more likely to be located at aromatic domains. Based on the results of XPS spectra, rather than to be oxidized by Cr(VI), carboxyl more tended to be complexed by chromium, which is regarded as the precondition for Cr(VI) reduction. Phenol, polysaccharide and methyl with distinct reaction activities successively acted as major electron donors for Cr(VI) reduction in different reaction stages. Consequently, it was determined that the retention of Cr(VI) by undissolved HA followed an adsorption-complexation-reduction mechanism, and based on this, a multi-step kinetic model with multiple types of complexation/reduction sites was developed to simulate the retention processes resulting in a much better fitting effect (R > 0.99) compared with traditional first-order and second-order kinetic models (R < 0.95). This demonstrated that the multi-step kinetic model is of great potential in accurately simulating the migration and transformation of Cr(VI) in soil environment.