Targeting glutamine metabolism has emerged as a promising therapeutic strategy in cancer, but the clinical development of 6-diazo-5-oxo-l-norleucine (DON) has been hindered by dose-limiting toxicities arising from its lack of selectivity. Here, we designed and synthesized mitochondria-directed, tumor-responsive DON prodrugs to enable controlled intracellular release and subcellular targeting. DON was conjugated to delocalized lipophilic cations via cleavable linkers, including glutathione (GSH)-responsive disulfide motifs, to selectively target mitochondrial glutaminase (GLS). Mito-DON-07 and Mito-DON-08 efficiently released DON in SK-Hep-1 cells and suppressed TCA cycle flux derived from 13C-glutamine, as demonstrated by stable isotope tracing. Both derivatives maintained antiproliferative activity comparable to DON in vitro while exhibiting improved tolerability in vivo. In an SK-Hep-1 xenograft model, Mito-DON-07 enhanced the antitumor efficacy of sorafenib relative to DON, with reduced tumor growth and increased apoptosis. In an MC38 allograft model, Mito-DON-08 induced more pronounced remodeling of the tumor immune microenvironment, including an increased effector-to-suppressor T cell ratio. Notably, Mito-DON derivatives showed reduced systemic toxicity compared with free DON. These findings establish mitochondria-targeted, GSH-responsive prodrug design as an effective strategy to improve the selectivity of glutamine metabolism inhibition and support combination-based cancer therapy.