Nephrotoxicity is an important problem during methotrexate (MTX) treatment, which has been widely used for the treatment of several cancer types. Females are less susceptible to kidney diseases; however, the reason for this condition has not to be fully clarified. But sex hormones such as estrogen may have a protective effect on the kidney. We aimed to evaluate the possible protective role of estrogen on the MTX-induced renal epithelial cell death. Primary renal proximal tubular epithelial cells (RPTEC) were incubated with MTX (1, 10 and 100M), either alone or in combination with the 17-estradiol, G protein-coupled estrogen receptor 1 (GPER1) agonist G-1, estrogen receptor alpha agonist propyl pyrazole triol (PPT), estrogen receptor beta agonist diarylpropionitrile (DPN). Cell viability was determined by MTT assays. Interleukin (IL)-1, IL-6, superoxide dismutase (SOD) and malondialdehyde (MDA) levels were determined in RPTEC. Approximately half of the cell death was observed with 10M MTX incubation for 48h. The cell death was prevented by co-incubating with17-estradiol, PPT and G-1. MTX was significantly induced IL-1 and IL-6.17-estradiol, PPT and G-1 significantly decreased effects of MTX. SOD activity was significantly decreased treatment with MTX compared to control group. SOD activity was increased with co-incubation with 17-estradioland G-1 compared to treatment with MTX. MDA levels significantly increased in treatment with MTX compared with the control group. Increased MDA levels by MTX-induced was decreased significantly by the treatment with 17-estradiol and G-1. These data indicate that especially 17-estradiol and G-1 may be useful in preventing undesirable effects of MTX in renal failure.