This study investigated the potential genes and related pathways in burn-induced myocardial injury. Rat myocardial injury induced by third-degree burn and the histopathological structures, apoptosis, and cardiac injury markers were then identified using hematoxylin & eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining, and enzyme-linked immunosorbent assay. Next, differentially expressed mRNAs were screened through next-generation sequencing (NGS), followed by functional annotation and key gene validation through quantitative reverse transcription-polymerase chain reaction. Subsequently, CD14 was screened out, and small interfering RNAs against CD14 were transfected to H9C2 cells to further verify the role of CD14 in burn-induced injury. The results showed that third-degree burn could markedly damage the structure of myocardial tissue, induce the apoptosis of myocardial cells, and increase the levels of myocardial injury-related markers, suggesting that burns could induce myocardial injury in rats. Besides, NGS data discovered that third-degree burn could result in 416 differentially upregulated mRNAs and 285 differentially downregulated mRNAs in myocardial tissue. It was also disclosed that differentially expressed mRNAs were mainly enriched in the phosphatidylinositol 3-kinase/Akt, mitogen-activated protein kinase (MAPK), and tumor necrosis factor signaling pathways. Furthermore, cell viability was significantly decreased in H9C2 cells treated with 10% rat burn serum. CD14 was significantly differentially expressed and screened out for further studies. Treatment with burn serum can significantly upregulate the phosphorylation level of extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase and the expression of cleaved caspase-3 and downregulate the expression of Bcl2 when compared with those in negative control of small interfering RNA transfected H9C2 cells, whereas interfering with CD14 expression reversed the effects of burn serum. The study demonstrated that burn serum treatment could activate the MAPK signaling pathway to promote cell apoptosis, and it can be reversed by interfering with the expression of CD14.