Selenium (Se) is an essential micronutrient for human and animal health. The Se status in a human population depends on the daily dietary Se intake, which is in part governed by the amount of Se available that enters the food chain from the soil. Applying cow manure is a traditional method to maintain or increase soil fertility for agricultural production. The application of cow manure affects the transformation of Se fraction in soil, which in turn affects its bioavailability. Until now, only a few studies have been conducted on the effects of cow manure on the bioavailability of Se in soil, and they have been limited to bulk soil. Moreover, the rhizosphere itself was also different from the bulk soil, which may have a significant effect on the fractionation, speciation and, eventually, the bioavailability of Se. Therefore, the objective of this study was to gain a better insight into how the application of manure amendments to selenium-contaminated soil affects the rhizosphere response of Indian mustard (Brassica juncea (L.) Czern. et Coss) and the speciation and the bioavailability of Se.