A series of Pt(IV) prodrugs containing a microtubule inhibitor paclitaxel (PTX) were synthesized and characterized and their antitumor activity was evaluated. Prodrugs 5–12 exhibited the most potent antitumor activity against the tested cancer lines, and simultaneously displayed lower toxicity toward the human normal cells MRC-5 and HL-7702 compared to mono- and co-therapy, respectively. As a representative, compound 5 (named Ptxplatin), conjugating cisplatin (CDDP) with one PTX, effectively entered the cancer cells in a time-dependent manner, significantly induced DNA damage, arrested the cell cycle at the G2/M stage, activated the expression of p53, and inhibited the migration of MCF-7 cells. Moreover, Ptxplatin induced mitochondrial dysfunction via the loss of the mitochondrial membrane potential (Δψ_m), increase of reactive oxygen species (ROS) content, promotion of Bax expression and inhibition of Bcl-2 expression. Besides, Ptxplatin induced excessive accumulation of intracellular ROS to trigger endoplasmic reticulum (ER) stress, leading to the significant exaltation of Ca²⁺ levels and upregulation of the ER-related protein PERK, ATF4 and CHOP. These findings indicated that Ptxplatin intervened in several cellular processes including the p53 apoptosis pathway, mitochondrial damage and ER stress to kill cancer cells.