Sepsis can cause myocardial injury, which is one of the leading causes of death in critically ill patients. The underlying mechanisms associated with sepsis-induced myocardial injury (SIMI) include impaired cardiac contractility, excessive cardiac inflammation, oxidative stress, cardiomyocyte apoptosis, and so on. SIMI is usually accompanied with dynamic changes of microRNAs (miRNAs) expression. And previous studies have shown that miR-214 plays a vital role in the protection of cardiomyocyte subjected to oxidative stress. In this study, we aimed to test whether miRNA-214 plays any roles in sepsis-induced myocardial injury. We performed quantitative real-time polymerase chain reaction in the septic mouse model induced by cecal ligation and puncture (CLP) and found the expression of miR-214 was upregulated. Then we transfected with the miRNA-214 precursor (pre-miR-214) to upregulate miR-214 expression and with the miR-214 inhibitor (anti-miR-214) to downregulate miR-214 expression respectively. Pre-miR-214 mouse model, anti-miR-214 mouse model, and wild-type mice were subjected to CLP or sham surgery. We observed that compared with control wild types, cardiac function, inflammatory response, the degree of myocardial injury, and myocyte apoptosis were remarkably alleviated in CLP-treated pre-miR-214 mice and aggravated in CLP-treated anti-miR-214 mice. Taken together, our study reveals that miR-214 has a protective effect in SIMI and thereby may provide a potential novel approach to treat SIMI.