Vandetanib is a novel multi-targeted tyrosine kinase inhibitor (TKI) that has demonstrated clinical efficacy in the long-term treatment of medullary thyroid carcinoma. However, its use is frequently associated with cardiovascular side effects, some of which may be life-threatening. Notably, multi-targeted TKIs have been increasingly implicated in the risk of cardiotoxicity. Among these, cardiac contractile dysfunction has emerged as a critical concern, yet the underlying mechanisms remain incompletely elucidated—particularly the phenomenon of excitation-contraction uncoupling. This study aims to investigate how vandetanib affects cardiac contractility, with a specific focus on sarcomeric protein degradation, and to identify potential molecular targets for reversing myofilament degradation. Chronic treatment with vandetanib led to a significant reduction in myocardial contractile function, the disruption of cardiac troponin T (cTnT), cardiac troponin I (cTnI) organization and downregulation of essential myofilament proteins, including cTnT (42.3% ± 5.6% protein loss)and MYOM1 (32.7% ± 4.9% protein loss), without notable suppression of calcium-handling proteins such as JPH2, p-PLN, and p-CaMKII. Calcium imaging revealed preserved sarcoplasmic reticulum (SR) calcium release, suggesting contractile dysfunction was not primarily calcium-driven. Instead, reduced myofilament calcium sensitivity and partial sarcomere disassembly were observed. Mechanistically, we identified upregulation of calpain expression and enzymatic overactivation as key mediators of sarcomeric protein degradation. Inhibition of calpain activity partially rescued vandetanib-induced loss of contractile proteins and preserved myofilament structure. Calpain1 overexpression aggravated, while calpain1 knockdown partially rescued, vandetanib-induced cTnT degradation. Our findings uncover a novel mechanism underlying TKI-induced cardiotoxicity, involving calpain-dependent degradation of cardiac myofilament proteins and independent of calcium dysregulation. This study highlights the critical role of sarcomere stability in maintaining cardiac function during TKI therapy and identifies calpain as a promising therapeutic target for cardioprotection, with calpain activation rather than calcium dysregulation being the key driver of vandetanib-induced cardiac dysfunction.