Chemotherapy resistance is the main reason of treatment failure in gastric cancer (GC). However, the mechanism of oxaliplatin (OXA) resistance remains unclear. Here, we demonstrate that extracellular mechanical signaling plays crucial roles in OXA resistance within GC. We selected OXA‐resistant GC patients and analyzed tumor tissues by single‐cell sequencing, and found that the mitochondrial content of GC cells increased in a biosynthesis‐independent manner. Moreover, we found that the increased mitochondria of GC cells were mainly derived from mesenchymal stromal cells (MSCs), which could repair the mitochondrial function and reduce the levels of mitophagy in GC cells, thus leading to OXA resistance. Furthermore, we investigated the underlying mechanism and found that mitochondrial transfer was mediated by mechanical signals of the extracellular matrix (ECM). After OXA administration, GC cells actively secreted ECM in the tumor microenvironment (TEM), increasing matrix stiffness of the tumor tissues, which promoted mitochondria to transfer from MSCs to GC cells via microvesicles (MVs). Meanwhile, inhibiting the mechanical‐related RhoA/ROCK1 pathway could alleviate OXA resistance in GC cells. In summary, these results indicate that matrix stiffness could be used as an indicator to identify chemotherapy resistance, and targeting mechanical‐related pathway could effectively alleviate OXA resistance and improve therapeutic efficacy.