B-cell lymphoma 6 (Bcl6) functions as a sequence-specific transcriptional repressor and negative regulator of many signaling proteins. The effects of Bcl6 on cardiomyocyte injury are not clear. This study was designed to determine whether Bcl6 affects hypoxia-induced cardiomyocyte injury and, if so, to identify the underlying mechanism. To meet this aim, cardiomyocytes were exposed to hypoxia and Bcl6 siRNA was used to silence Bcl6 in cardiomyocytes. Bcl6 knockdown under physiological conditions caused increased oxidative stress, apoptosis, and expression of pro-inflammatory cytokines. Increased inflammatory response, oxidative stress, and apoptosis were observed after cells were exposed to hypoxia for 24 h. Bcl6 knockdown aggravated cardiomyocyte injury when exposed to hypoxia. Bcl6 knockdown increased P38 activation without affecting JNK and ERK phosphorylation levels. Treatment with a P38 inhibitor reversed the Bcl6 silencing-induced deteriorating phenotype, as evidenced by reduced inflammatory response, improved oxidative stress response, and increased cell viability. The results indicate that Bcl6 knockdown causes cardiomyocyte injury at baseline conditions and aggravates cardiomyocyte hypoxia injury via activating the P38 pathway.