Multidrug resistance (MDR) is considered as a major obstacle in achieving an effective treatment of breast cancer. Paclitaxel has been used to treat cancers of the cervical, breast, ovarian and brain but MDR limits its therapeutic potential. Phytochemicals have received much interest in recent decades especially in combination approaches to tackle MDR due to their negligible harm to healthy cells and synergistic potential. Considering this notion, the present study aimed at investigating the synergistic activity of 4-MTBITC and PTX against a panel of breast cancer cells. Our results revealed that the combination had a significant antiproliferative activity against T-47D cells. Mechanistic studies revealed that 4-MTBITC and PTX also promoted the production of reactive oxygen species (ROS) and reduced mitochondrial membrane potential. In the presence of 4-MTBITC- PTX, T-47D cells were found to be arrested in the G2/M phase which also confirmed the enhancement of late apoptotic cell population in the flow cytometer analysis. In western blot experiment, the combination had a significant decrease in Bcl-xl protein level, whereas a higher level of p53, cleaved caspase-3, and cleaved caspase-9 proteins compared to individual treatment in T-47D cells. The RT-qPCR analysis also showed that the combination had significant upregulation in the gene expression of p53, cytochrome-c, Apaf-1 and downregulation in the expression of Bcl-2 gene in T-47D cells. Hence, all the results showed that a combination of 4-MTBITC-PTX significantly enhanced the apoptosis pathway in the T-47D cell line which indicates its clinical application for the treatment of breast cancer.Abbreviations: Apaf-1: Apoptotic protease activating factor 1; AO/EB: Acridine orange/ethidium bromide; Bcl-2: B-cell lymphoma 2; CI: Combination Index; Cyt-c: Cytochrome c; CO2: Carbon dioxide; DCFH-DA 2,7-Dichloroflourescein diacetate; DMEM: Dulbecco's modified Eagle's medium; ELISA: Enzyme-linked immunosorbent assay; EA: Early apoptosis; EDTA: Ethylenediaminetetraacetic acid; L929: Normal mouse fibroblast cells; LA: Late apoptosis; L: Live; 4-MTBITC: 4-methylthiobutyl isothiocyanate; MCF-7: Human breast cancer cells; MDA-MB-231: Human triple negative breast cancer cells; MMP: Mitochondria membrane potential; MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide; NCCS: National Centre for Cell Science; N: Necrotic; PTX Paclitaxel; PVDF: Polyvinylidene fluoride; PAGE: Polyacrylamide gel electrophoresis; PBS: Phosphate-buffered saline; RPMI-1640: Roswell Park Memorial Institute Medium- 1640; RT-qPCR: Quantitative real-time polymerase chain reaction; ROS: Reactive oxygen species; Rh-123: Rhodamine123; g Relative centrifugal force; SDS: Sodium dodecyl sulphate; SEM: Scanning electron microscopy; T-47D: Human estrogen positive breast cancer cells; WB: Western blotting.