The bioavailability of beta-carotene is highly variable and largely depends on environmental conditions and interactions with other compounds present in the food matrix. Dietary fibers can have a significant impact on bioavailability due to the possibility of formation of complex compounds with beta-carotene and other interactions. The basic hypothesis of this work was that the way dietary fibers interact with beta-carotene (and the consequent effect on bioavailability) significantly depends on the physico-chemical properties of dietary fibers. For this reason, the binding capacity of different solubility (for beta-carotene) and the influence of fiber mixtures (with different ratios of soluble and insoluble fibers) on the in vitro bioavailability of cis- and trans- isomers of beta carotene from the real food matrix were investigated in this work. The bioavailability of beta-carotene was determined by in vitro static digestion simulation according to a standardized method, and beta-carotene concentrations were determined spectrophotometrically, i.e. using high-resolution liquid chromatography (HPLC). The results showed that the physico-chemical properties of dietary fibers significantly affect the ability to bind betacarotene, with insoluble cellulose-type fibers having the most pronounced negative effects. Dietary fiber mixtures with a predominantly insoluble component have a negative effect on the in vitro bioavailability of beta-carotene, while this effect is absent in mixtures consisting of predominantly soluble fibers. The effect is more pronounced on the transisomers of beta-carotene. The above results significantly contribute to the very limited knowledge about the influence of dietary fibers on the bioavailability of beta-carotene and are particularly relevant in the context of the smart design of functional food that requires a high content of dietary fiber while maintaining satisfactory bioavailability of bioactive components such as beta-carotene.