This study aims to explore the effect and mechanism of pyruvate kinase M2 (PKM2) on chemotherapy resistance of estrogen receptor-positive breast cancer (ER+ BC) by regulating aerobic glycolysis. The expression of PKM2 in ER+ BC MCF-7 cells, T47D cells and MCF-7/ADR cells (which are subject to adriamycin/ADR induction) were determined by quantitative real-time PCR and western blot. MCF-7/ADR (M/A) cells were grouped into blank group (M/A), negative group (M/A+NC), low expression of PKM2 group (M/A+si-PKM2 group), overexpression of PKM2 group (M/A+PKM2 group) and glycolysis inhibition group (M/A+PKM2+2-DG group). Quantitative real-time PCR and western blot were applied to measure the expressions of PKM2, multidrug resistance, and glutathione-S-transferase π. Glucose and lactic acid kit was used to detect the amount of glucose uptake and lactic production. Cell variability, clone formation ability, and cell apoptosis were respectively measured by MTT, clone formation assay, and flow cytometry. Transwell assay and scratch assay were applied for cell invasion and migration ability. By overexpressing PKM2 in MCF-7 and T47D cells and using 2-DG, the effect on sensitivity of adriamycin amycin was explored. MCF-7/ADR cells have both elevated mRNA and protein expressions of PKM2 when compared with MCF-7 cells (both P<0.05). The cell activity of the M/A+si-PKM2+ADR group was notably lower than that in the M/A+ADR group and M/A+NC+ADR group (both P<0.05). In the M/A+si-PKM2 group, expressions of PKM2, multidrug resistance, and glutathione-S-transferase π, along with the amount of glucose uptake and lactic production, as well as cell variability, clone formation ability, and cell invasion and migration ability were inhibited, whereas cell apoptosis was increased in comparison with the M/A group and M/A+NC group (all P<0.05). On comparing with both the M/A group and the M/A+NC group, the M/A+si-PKM2 group displayed contrary tendency with the M/A+PKM2 group. The M/A+PKM2+2-DG group had elevated PKM2 expression compared with the M/A group and the M/A+NC group (all P<0.05). In MCF-7 and T47D cells with overexpression of PKM2, the sensitivity to adriamycin amycin, and cell apoptosis were suppressed, whereas the clone formation, invasion, and migration ability were enhanced. After 2-DG, the sensitivity on MCF-7 and T47D cells was enhanced while clone formation, invasion, migration and cell apoptosis rate were decreased (all P<0.05). PKM2 enhances chemotherapy resistance on ER+ BC by promoting aerobic glycolysis.