Secretory granules released by cytotoxic T lymphocytes (CTLs) are powerful weapons against intracellular microbes and tumor cells. Despite significant progress， there is still limited information on the molecular mechanisms implicated in target-driven degranulation， effector cell survival and composition and structure of the lytic granules. Here， using a proteomic approach we identified a panel of putative cytotoxic granule proteins， including some already known granule constituents and novel proteins that contribute to regulate the CTL lytic machinery. Particularly， we identified galectin-1 (Gal1)， an endogenous immune regulatory lectin， as an integral component of the secretory granule machinery and unveil the unexpected function of this lectin in regulating CTL killing activity. Mechanistic studies revealed the ability of Gal1 to control the non-secretory lytic pathway by influencing Fas-Fas ligand interactions. This study offers new insights on the composition of the cytotoxic granule machinery， highlighting the dynamic cross talk between secretory and non-secretory pathways in controlling CTL lytic function.