Intestinal enteroids are “mini-guts” that recapitulate essential features of the intestinal epithelium in vitro. We developed and characterized native basal-out (BO) and morphogenically everted apical-out (AO) jejunal enteroids cultured in suspension. Using quantitative proteomics, we show that these enteroids progressively adopt protein profiles closely resembling those of freshly isolated villus enterocytes and human jejunal tissue.