Summary. Background: Altered Ca2+ homeostasis contributes significantly to platelet hyperactivity in diabetes mellitus. Calsequestrin (CSQ), as a Ca2+ buffer protein in the sarcoplasmic reticulum, also regulates the Ca2+ release process in muscles. We hypothesized that CSQ may be expressed in platelets, but is altered and involved in diabetic platelet Ca2+ abnormalities and hyperaggregability. Methods: CSQ expression in platelets from streptozotocin-induced type 1 diabetes rats, type 2 diabetes volunteers and Goto-Kakizaki rats were analyzed by western blotting and RT-qPCR. Platelet Ca2+ and aggregation were evaluated with Fura2 and an aggregometer, respectively. Results: Platelets from diabetic patients and rats exhibited increased resting Ca2+ levels, and hyperactive Ca2+ and aggregation responses to agonists. This enhanced basal Ca2+ was largely dependent on intracellular Ca2+ and insensitive to inositol 1,4,5-trisphosphate receptor (IP3R) antagonism. Additionally, the expression of the skeletal CSQ isotype (CSQ-1) was detected in both rat and human platelets, but its levels were significantly lowered in diabetic platelets as compared with normal platelets. Impairment of CSQ by trifluoperazine caused concentration-dependent Ca2+ release in normal platelets and HEK293 cells. Knocking down CSQ-1 in HEK293 cells resulted in increased leakage of Ca2+, which was also insensitive to IP3R inhibition, and exaggerated Ca2+ release following carbachol treatment. Conclusions: Downregulation of CSQ-1 in diabetic platelets and impairment of CSQ-1 in normal cells leads to disturbed Ca2+ release, demonstrating a potential role for CSQ-1 in the regulation of the platelet Ca2+ release process and a possible causal contribution to diabetic platelet hyperactivity.