Heme oxygenase 1 (HO-1) inhibits endothelial cell (EC) apoptosis by generating carbon monoxide (CO). This study demonstrates that HO-1 prevents EC apoptosis through CO production, which activates the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Overexpression of HO-1 or induction by heme protects ECs from apoptosis, while blocking HO-1 activity or CO action with tin protoporphyrin (SnPPIX) or hemoglobin (Hb) abrogates this protection. Exogenous CO, under HO-1 inhibition, can substitute for HO-1 in preventing EC apoptosis. The antiapoptotic effect of HO-1 is mediated by CO, which activates p38 MAPK. This activation is dependent on TNF-α and is inhibited by p38 MAPK inhibitors like SB203580. Additionally, CO and iron chelation (via DFO) have additive effects in suppressing EC apoptosis. The study also shows that HO-1 can protect ECs that do not express HO-1 by releasing CO. The antiapoptotic effect of HO-1 is not mediated by guanylyl cyclase or cGMP generation. These findings highlight the role of CO as a signaling molecule in EC protection and the importance of HO-1 in preventing EC apoptosis through CO production.Heme oxygenase 1 (HO-1) inhibits endothelial cell (EC) apoptosis by generating carbon monoxide (CO). This study demonstrates that HO-1 prevents EC apoptosis through CO production, which activates the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Overexpression of HO-1 or induction by heme protects ECs from apoptosis, while blocking HO-1 activity or CO action with tin protoporphyrin (SnPPIX) or hemoglobin (Hb) abrogates this protection. Exogenous CO, under HO-1 inhibition, can substitute for HO-1 in preventing EC apoptosis. The antiapoptotic effect of HO-1 is mediated by CO, which activates p38 MAPK. This activation is dependent on TNF-α and is inhibited by p38 MAPK inhibitors like SB203580. Additionally, CO and iron chelation (via DFO) have additive effects in suppressing EC apoptosis. The study also shows that HO-1 can protect ECs that do not express HO-1 by releasing CO. The antiapoptotic effect of HO-1 is not mediated by guanylyl cyclase or cGMP generation. These findings highlight the role of CO as a signaling molecule in EC protection and the importance of HO-1 in preventing EC apoptosis through CO production.