Chloroplast lipids are synthesized via two distinct pathways: the plastidic pathway and endoplasmic reticulum (ER) pathway. We previously reported that the contribution of the two pathways toward chloroplast development is different between mesophyll cells and guard cells in Arabidopsis leaf tissues, and that the ER pathway plays a major role in guard cell chloroplast development. However, little is known about the contribution of the two pathways toward chloroplast development in other tissue cells, and in this study, we focused on the root cells. Chloroplast development is normally repressed in roots but can be induced when the roots are detached from the shoots (root greening). We found that similar to guard cells, root cells exhibit a higher proportion of glycolipid from the ER pathway. Root greening was repressed in the gles1 mutant, which has a defect in ER-to-plastid lipid transportation via the ER pathway, while normal root greening was observed in the ats1 mutant, whose plastidic pathway is blocked. Lipid analysis revealed that the gles1 mutation caused drastic decrease in the ER-derived glycolipids in roots. Furthermore, the gles1 detached roots showed smaller chloroplasts containing little starch than WT. These results suggest that the ER pathway has a significant contribution toward chloroplast development in the root cells.