Various characterization techniques, including UV-Vis, FTIR, EPR and XPS, were used for the description of structural changes in Fe-silicalite with low concentration of iron, which was activated using different methods (by conventional hydrothermal pretreatment at 600 °C and by treatment in the diluted flow of ammonia and propane in helium at 540 °C). Activated Fe-silicalite was subsequently used as a catalyst for the direct ammoxidation of propane. It has been shown that the studied methods of activation lead to materials with different catalytic behavior (activation in the flow of ammonia and propane resulting in the more active and selective catalytic materials). Results from the catalytic test are further discussed together with the results from the characterization of activated samples.