Eukaryotic initiation factor 3a (eIF3a) has been suggested to play a regulatory role in mRNA translation. Decreased eIF3a expression has been observed in differentiated cells while higher levels have been observed in cancer cells. However, whether eIF3a plays any role in differentiation and development is currently unknown. Here, we investigated eIF3a expression during mouse development and its role in differentiation of colon epithelial cells. We found that eIF3a expression was higher in fetal tissues compared with postnatal ones. Its expression in intestine, stomach, and lung abruptly stopped on the 18th day in gestation but persisted in liver, kidney, and heart throughout the postnatal stage at decreased levels. Similarly, eIF3a expression in colon cancer cell lines, HT-29 and Caco-2, drastically decreased prior to differentiation. Enforced eIF3a expression inhibited while knocking it down using small interference RNA promoted Caco-2 differentiation. Thus, eIF3a may play some roles in development and differentiation and that the decreased eIF3a expression may be a pre-requisite of intestinal epithelial cell differentiation.