Abstract Lipopolysaccharide (LPS) induces cyclooxygenase-2 (COX-2) expression in cardiomyocytes, which plays a role in myocardial depression during endotoxemia. The purpose of this study was to investigate the role of phosphatidylinositol (PI)-phospholipase Cγ1 (PLCγ1) in cardiac COX-2 expression in vitro and in vivo. In cultured mouse neonatal cardiomyocytes, LPS increased PLCγ1 phosphorylation and COX-2 expression. Knockdown of PLCγ1 with specific siRNA or inhibition of PI-PLC with U73122 attenuated COX-2 mRNA and protein expression induced by LPS (1 μg/ml). PLCγ1 activation by LPS also increased ERK1/2 MAPK phosphorylation, and inhibition of ERK1/2 MAPK blocked the effect of PLCγ1 on COX-2 expression. Furthermore, activation of PLCγ1 is a consequence of the Src family activation since inhibition of Src abrogated whereas over-expression of Src enhanced PLCγ1 phosphorylation and COX-2 expression in LPS-stimulated cardiomyocytes. To investigate the role of PLCγ1 in endotoxemia, wild-type and PLCγ1 +/− adult mice were pre-treated with U73122, or its inactive analog, U73343 (9 mg/kg, i.p.), or vehicle for 15 min followed by LPS (4 mg/kg, i.p.) for 4 h. U73122 or heterozygous deletion of PLCγ1 decreased cardiac COX-2 expression. The phosphorylation of ERK1/2 MAPK induced by LPS was also attenuated in U73122- or PLCγ1 +/− compared to U73343-treated or wild-type littermate hearts, respectively. In conclusion, our study suggests that PLCγ1 signalling represents a novel pathway regulating cardiac COX-2 expression during LPS stimulation. The Src family is responsible for PLCγ1 activation, which signals the ERK1/2 MAPK pathway, resulting in COX-2 production in LPS-stimulated cardiomyocytes.