Trastuzumab (TRZ) is a standard drug for the treatment of HER-2-positive breast cancer, but its cardiotoxicity seriously affects the prognosis of patients, and the potential mechanism of TRZ-induced cardiomyocyte atrophy leading to cardiotoxicity remains unclear. This study aimed to investigate the potential targets and signaling pathways of TRZ-induced cardiotoxicity using single-cell transcriptome sequencing (scRNA-seq) and bioinformatics methods. A total of 618 differentially expressed genes (DEGs) were identified through scRNA-seq and bioinformatics analysis. Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, Reactome analysis, Wiki pathway analysis, Protein-Protein Interaction (PPI) network analysis, and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) results indicated that immune cell-derived FN1 may play a pivotal role in TRZ-induced cardiotoxicity by activating PI3K/AKT-mediated mTOR-independent signaling pathways in cardiomyocytes through extracellular matrix (ECM) mechanisms. Western blot analysis revealed that the expression of FN1, p-PI3K/PI3K, and p-AKT/AKT proteins was elevated in the TRZ group, no elevation of p-mTOR/mTOR and the autophagy level of cardiomyocytes was elevated, as well as the levels of myocardial atrophy and apoptosis were enhanced after treatment with TRZ. TEM revealed increased autolysosomes in TRZ-treated samples, while immunofluorescence analysis demonstrated exacerbated myocardial injury and significant cardiomyocyte atrophy in the TRZ group compared to CON. Our results demonstrate that TRZ-primed immune cells release FN1 which, through ECM triggers non-canonical (mTOR-independent) PI3K/AKT signaling in cardiomyocytes. This aberrant pathway drives excessive autophagy, initiating a pathological cascade from cellular atrophy to apoptotic death and ultimately manifesting as clinical cardiotoxicity.