Loading drug-maleimide (MAL) conjugates into liposomes preloaded with glutathione (GSH) can prepare the liposome encapsulating GSH-conjugated prodrugs, which serve as a feasible way to construct liposomal formulation. However, the effects ofthelinker on the development of this liposomal system remained unclear. Herein, docetaxel (DTX)-MAL conjugates linked by various linkers were used for such studies. It was found that the linker influenced the aqueous solubility of DTX-MALs, which further affected their loading efficiency in liposomes. The linker significantly influenced the DTX release from liposomal DTX-GSH (DTX-LIPs) by impacting the activation rate of DTX-GSH outside liposomes. Notably, DTX-LIPs containing the rapidly-activated DTX-GSH exhibited much more potent antitumor activity in the 4T1 breast cancer xenograft than other DTX-LIPs and commercial DTX injections. Analysis of the drug release mechanism revealed that DTX-GSH was first released from theliposome and consequently activated into DTX, and the prodrug activation was the rate-limiting step for DTX release. These results highlighted the crucial role of linkers in the drug loading, drug release, and antitumor activity of DTX-LIPs prepared by active click loading, which would effectively guide the rational design of liposomal drugs for improved antitumor efficacy.