he killing efficiency of cytotoxic T lymphocytes (CTLs) is tightly regulated by intracellular Ca2+ concentration. Glucose is the key energy source for CTLs, lack of which significantly impairs CTL activation, proliferation and effector functions. The impact of high glucose on Ca2+ influx in CTLs remains largely elusive. In this work, we stimulated primary human CD8+ T cells in medium containing either 25 mM (high glucose, HG) or 5.6 mM glucose (normal glucose, NG). We found that store‐operated calcium entry (SOCE) induced by thapsigargin (Tg) is elevated in HG‐cultured CTLs compared to their counterparts in NG. Unexpectedly, the Ca2+ influx elicited by recognition of target cells is reduced in HG‐cultured CTLs. Under HG condition, STIM1 and STIM2, the calcium sensors in the endoplasmic reticulum (ER), were down‐regulated; ORAI1, the main structural component of calcium‐release activated channels, remained unchanged, whereas ORAI2 and ORAI3 were up‐regulated. The fraction of necrosis of HG‐cultured CTLs was enhanced after killing without affecting glucose uptake. Thus, our findings reveal that HG has a distinctive impact on Tg‐evoked SOCE and target recognition‐induced Ca2+ influx in CTLs and causes more CTL death after killing, suggesting a novel regulatory role of high glucose on modulating CTL functions.