We previously reported that hydrogen sulfide (H2S) preconditioning (SP) produces cardioprotection in isolated rat cardiomyocytes. The present study was designed to determine the involvement of cyclooxygenase-2 (COX-2) in the SP-induced delayed cardioprotection. Isolated cardiac myocytes were treated with NaHS (100 μM, a H2S donor) for 30 min and then cultured for 20 h followed by ischemia/reperfusion insults. SP significantly increased cell viability, percentage of rod-shaped cells, and myocyte contractility after 10 min of reperfusion. Given 30 min before and during lethal ischemia, two selective COX-2 inhibitors, NS-398 and celebrex, abrogated SP-induced cardioprotective effects. Moreover, SP upregulated the expression of COX-2 and increased PGE2 production in the cardiac myocytes. These effects were significantly attenuated by glibenclamide, an ATP-sensitive K+ channel (KATP) blocker, and chelerythrine, a selective protein kinase C (PKC) inhibitor, suggesting that activation of both KATP and PKC is required for the stimulation of COX-2. Additionally, NG-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor, failed to regulate COX-2 protein expression but inhibited SP-enhanced COX-2 activity and PGE2 production. In conclusion, we provided the first evidence that SP may produce delayed cardioprotection via KATP/PKC dependent induction of COX-2 expression and via nitric oxide-induced COX-2 activation.