Selective separation of vitamin C from the mixture with 2-ketogluconic acid (the main byproduct from the fermentation process) by reactive extraction using Amber lite LA-2 dissolved in dichloromethane has been analyzed. The mechanism of the interfacial reaction pointed out that, indifferent of the pH value, only one molecule of vitamin C or 2-ketogluconic acid and one of extractant react at the interface, the resulting interfacial product being a hydrophobic ammonium salt. The obtained results indicated that it is possible to separate vitamin C by modifying the aqueous phase pH.