Hydrophobic bioactives can be more easily incorporated into food and have their bioavailability enhanced if nanostructured lipid carriers (NLC) are used as carriers. In the present study, beta-carotene-loaded NLC were produced by low emulsification using murumuru butter and a mixture of Span 80 and Cremophor RH40 as surfactants. Their average diameter was 35 nm and alpha-tocopherol was required to protect the encapsulated β-carotene. Besides the evaluation of their physicochemical stability, NLC were submitted to dynamic in vitro digestion and cell viability assays with Caco-2 and HEPG cells. The bioaccessibility of beta-carotene in the dynamic system was about 42%. Regarding cell viability, results indicated NLC were toxic to the cell cultures tested. Such high toxicity is probably related to the type of surfactant used and to the extremely reduced particle size, which may have led to an intense and fast permeation of the NLC through the cells.