To increase the intestinal delivery of dietary β-carotene, there is a need to develop nanostructured food systems to encapsulate this fat soluble bioactive. The aim of this study was to evaluate the bioacessibility and bioavailability across the intestinal barrier of β-carotene-enriched nanoemulsions stabilised with two emulsifiers (lecithin or sodium caseinate) by coupling an in vitro gastrointestinal digestion with two in vitro cell culture models (Caco-2 or co-culture of Caco-2/HT29-MTX). Nanoemulsions stabilised with lecithin had significantly higher β-carotene in the gastrointestinal digested micellar fraction, lower β-carotene in the Caco-2 (and Caco-2/HT29-MTX) apical compartment and significantly higher β-carotene in Caco-2 cellular content compared to β-carotene-enriched nanoemulsions stabilised with sodium caseinate. Finally, to assess anti-inflammatory activity of digested nanoemulsions, lipopolysaccharide stimulated macrophages were exposed to Caco- 2 basolateral samples with levels of TNF-α and IL-β, subsequently quantified. A TNF-α response from stimulated THP-1 macrophages was elicited by basolateral samples, regardless the emulsifier used to formulate nanoemulsions. This study demonstrated that β-carotene permeability is influenced by the food derived emulsifier used for stabilising nanoemulsions, indicating that composition may be a critical factor for β-carotene delivery.