P-glycoprotein (P-gp, ABCB1 member of the ABC (ATP-binding cassette) transporter family) localized in leukemia cell plasma membranes is known to reduce cell sensitivity to a large but well-defined group of chemicals known as P-gp substrates. However, we found previously that P-gp-positive sublines of L1210 murine leukemia cells (R and T) but not parental P-gp-negative parental cells (S) are resistant to the endoplasmic reticulum (ER) stressor tunicamycin (an N-glycosylation inhibitor). Here, we elucidated the mechanism of tunicamycin resistance in P-gp-positive cells. We found that tunicamycin at a sublethal concentration of 0.1 µM induced retention of the cells in the G1 phase of the cell cycle only in the P-gp negative variant of L1210 cells. P-gp-positive L1210 cell variants had higher expression of the ER stress chaperone GRP78/BiP compared to that of P-gp-negative cells, in which tunicamycin induced larger upregulation of CHOP (C/EBP homologous protein). Transfection of the sensitive P-gp-negative cells with plasmids containing GRP78/BiP antagonized tunicamycin-induced CHOP expression and reduced tunicamycin-induced arrest of cells in the G1 phase of the cell cycle. Taken together, these data suggest that the resistance of P-gp-positive cells to tunicamycin is due to increased levels of GRP78/BiP, which is overexpressed in both resistant variants of L1210 cells.