<p>The aim of this study was to explore the effect of dexmedetomidine in combination with sevoflurane pretreatment on the myocardial Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor (NF)-κB signaling pathway in patients undergoing open-heart surgery under cardiopulmonary bypass (CPB). A total of 92 patients who underwent open-heart surgery under CPB in the hospital were retrospectively analyzed and divided into an observation group (<i>n</i> = 46) and a control group (<i>n</i> = 46) according to the different methods of pretreatment; the observation group was pretreated with dexmedetomidine in combination with sevoflurane, and the control group was given an equal volume of normal saline. Both groups exhibited higher positive expression rates of TLR4 and NF-κB and fluorescence intensity of MyD88 at T1, T2, and T3 than at T0 (<i>P</i> &lt; 0.05); the positive expression rates of TLR4 and NF-κB and fluorescence intensity of MyD88 at T1, T2, and T3 in the observation group were significantly lower than those in the control group (<i>P</i> &lt; 0.05). The levels of TNF-α, IL-6, and IL-8 were higher at T1, T2, and T3 than at T0 in both groups, and were lower in the observation group than in the control group (<i>P</i> &lt; 0.05). The levels of SOD at T1, T2, and T3 were lower than those at T0 in both groups and were higher in the observation group, whereas the levels of MDA and HO-1 at T1, T2, and T3 were higher than those at T0 and were lower in the observation group (<i>P</i> &lt; 0.05). The levels of CK-MB, cTnI, and CK at T1, T2, and T3 were higher than those at T0 in both groups, and were lower in the observation group (<i>P</i> &lt; 0.05). The levels of HR, MAP, and SaO2 at T1 and T2 in the observation group were higher than those in the control group (<i>P</i> &lt; 0.05). Thus, dexmedetomidine in combination with sevoflurane pretreatment can inhibit the TLR4/MyD88/NF-κB signaling pathway and alleviate inflammatory responses and oxidative stress levels.</p>