Abstract: AIM To determine the effect of thioredoxin-interacting protein (TXNIP) mediated NLRP3 inflammasome activation on cardiac microvascular endothelial cells (CMECs) during hypoxia/reoxygenation (H/R) injury. METHODS CMECs isolated from the hearts of adult mice were randomized into control group, H/R group, H/R+scrambled siRNA group and H/R+TXNIP siRNA group. Levels of IL-1β were detected by ELISA kits. The interaction between TXNIP and NLRP3 was determined by immunoprecipitation. Expressions of TXNIP and NLRP3 were analyzed by Western blot, and caspase-3 activity and LDH release were assessed with test kits. RESULTS H/R increased NLRP3 expression (P<0.05), IL-1β level (P<0.01) and the interaction between TXNIP and NLRP3 (P<0.01) in CMECs compared with control group. Blocking TXNIP signaling with siRNA significantly inhibited NLRP3 activation (P<0.05) and alleviated endothelial injury evidenced by increased caspase-3 activity LDH release (P<0.05). CONCLUSION H/R injury promotes NLRP3 activation and increases the interaction between TXNIP and NLRP3. Blocking TXNIP signaling reduces NLRP3 activation and endothelial injury. TXNIP mediated NLRP3 activation may be a novel mechanism in H/R injury of CMECs.