Organoids are stem-cell-derived 3D multicellular structures that reproduce the characteristics of the tissue of origin much better than long-term cultured immortalized cell lines. Patient-derived organoids (PDO) are increasingly used to dissect the tumorigenesis process and for personalized drug screenings in colorectal cancer (CRC). Many epidemiological and preclinical studies performed using carcinoma cell lines, cancer-associated fibroblasts, and mouse models support a protective action of vitamin D/calcitriol against CRC. Here, we have studied the effect of calcitriol on cell differentiation in colon PDO generated by healthy tissue epithelial stem cells (normal PDO) or by cancer stem cells (tumor PDO). We optimized PDO culture conditions to differentiate normal colon stem cells toward absorptive enterocytes and goblet cells. Differentiation was assessed by gene and protein expression assays (RT-qPCR, RNA-seq, immunofluorescence and western blot) and ultrastructural electron microscopy analysis of cell phenotype. Bone morphogenetic protein (BMP)4 promoted enterocytic differentiation with increased expression of absorptive markers and formation of surface microvilli. The Notch pathway inhibitor DBZ caused goblet cell differentiation with high expression of mucosecretory markers and intracellular accumulation of mucus-containing secretory vesicles. The combination of BMP4 and DBZ lead to the strong downregulation of stemness genes and the balanced induction of both cell lineage markers, enterocytes and goblet cells. Interestingly, calcitriol reduced the expression of the differentiation markers at RNA and protein levels and, concordantly, diminished the percentage of cells showing a highly differentiated phenotype. In contrast, we found that tumor PDO organoids responded poorly to BMP4 and were unresponsive to DBZ, with only a subset of cells showing some partial differentiation features. This behavior most probably relies on the mutational load that causes the aberrant activation of the canonical WNT and the RAS-BRAF pathways in CRC patients. In tumor PDO, calcitriol treatment partially potentiated the effect of BMP4 increasing the number of cells displaying still immature enterocytic differentiation features. In summary, we have stablished a new differentiation model in colon normal PDO that could be useful to study the effects of different agents on healthy and disease human colon epithelium. We have also shown that calcitriol favors the stemness of normal colon stem cells, which may prevent the exhaustion of this critical cell population in aging and alleviate damage of intestinal epithelium linked to inflammatory bowel diseases and chemotherapy.The pro-differentiation effect of calcitriol on colon tumor PDO, though limited, may add to its reported antitumor effects on carcinoma, stromal and immune cells.