Natural aluminosilicates are promising sorbents that can be used to separate and isolate biologically active substances, namely vitamins. Hydrochloride pyridoxine (vitamin B6) is an important biologically active compound necessary for the normal functioning of the body. This article describes the investigation of absorption regularities of pyridoxine hydrochloride on the glauconite. Clay aluminosilicate, i.e. glauconite with a layered structure (from south-west of the Voronezh anteclise) was chosen as the subject for the study. Its phase composition is represented by mixed-layer minerals of illite-smectite with 20:80 ratio of layers and a high iron level. Its interlayer space has water molecules and K+, Mg2+ and Ca2+ exchangeable cations. The study of equilibrium in glauconite (0.02–0.06 mm fraction), i.e. aqueous solution of vitamin B6, was carried out at a temperature of 295 K under static conditions by using variable concentrations. The interval for the vitamin concentrations within 0.02 and 7.25 mmol/dm3 in the pH solution was 3.50–3.94, where pyridoxine hydrochloride was mainly present in the solution in the form of cations. Absorption regularities of pyridoxine hydrochloride on the glauconite were identifi ed and explained. To conduct a quantitative description of sorption equilibrium in the interval of concentrations that lead to the formation of the vitamin monolayer the linearized equations of Langmuir, Freundlich, Redlich–Peterson models were used. Whereas the Brunauer–Emmett–Teller (BET) theory was used to describe the isotherm as a whole. It was established that the sorption process involves the localization of pyridoxine cations at the negatively charged centres of glauconite which results from ion exchanges with extra framework cations and the formation of associates. Pyridoxine sorption caused by ion exchange can be described with high probability by the Langmuir model. The multi-molecular nature of the sorption and the increase in the solution concentration determine the formation of pyridoxine associates caused by the formation of hydrogen bonds and π-π interactions in the pyridine ring. The equilibrium coeffi cient for the pyridoxine distribution in the glauconite structure was calculated. The increase in concentration of the solution leads to the sharp decrease in the formation of the monolayer. The polymolecular sorption of the vitamin is characterized by a slight change in the distribution coeffi cient.