Cytotoxic T lymphocytes (CTLs) are the key players to eliminate tumor cells. In solid tumors, dense extracellular matrix (ECM) serves as physical barriers to hinder infiltration and dampen functions of CTLs. However, how the killing capacity of T cells is regulated by dense matrices still remains largely unknown. In this work, we analyzed functional changes of primary human CTLs in dense matrices and the underlying mechanisms. More specifically, among all killing related processes, only CTL migration was reduced in dense matrices, leading to impaired killing capacity. Both the pore size and stiffness of the matrices influence CTL migration. The microtubule‐network is a negative regulator for CTL migration in dense collagen matrices. Perturbing microtubule integrity by nocodazole or vinblastine (a chemotherapeutic agent) substantially enhanced killing efficiency of CTLs in dense matrices. Our findings will inspire new strategies for tumor treatment, for example combining microtubule‐targeting chemotherapeutic agents with CTL adoptive immunotherapy to treat solid tumors.