Accumulating evidence has reported the role of microRNA-21-3p (miR-21-3p) in sepsis, and our objective was to discuss the effect of the miR-21-3p/regulator of calcineurin 1 (Rcan1) axis on cardiomyocyte apoptosis of septic rats. miR-21-3p and Rcan1 expression in myocardial tissues of lipopolysaccharide (LPS)-treated rats and LPS-treated H9c2 cardiomyocytes was determined. The influences of downregulating miR-21-3p or upregulating Rcan1 in cardiomyocyte apoptosis of LPS-treated rats and LPS-treated H9c2 cardiomyocytes were then evaluated. The target relation between Rcan1 and miR-21-3p was verified. It was observed that miR-21-3p was elevated and Rcan1 was reduced in LPS-treated rats and LPS-treated H9c2 cardiomyocytes. Downregulating miR-21-3p or upregulating Rcan1 could suppress cardiomyocyte apoptosis of LPS-treated rats and LPS-treated H9c2 cardiomyocytes. Based on the dual-luciferase activity assay, miR-21-3p was directly targeted Rcan1 in H9c2 cardiomyocytes. In the rescue experiment, the LPS+miR-21-3p inhibitor+si-Rcan1 group enhanced the apoptosis of H9c2 cardiomyocytes in comparison to the LPS+miR-21-3p inhibitor+si-NC group. Together, our findings identify that the miR-21-3p/Rcan1 axis may affect apoptosis of cardiomyocytes in sepsis, which provides a new idea for understanding the potential mechanism of sepsis.