The development of resistance to trastuzumab in HER2-positive breast cancer is a serious clinical problem that limits the effectiveness of targeted therapy. In a significant proportion of patients, the mechanisms in the development of resistance remain poorly understood. The BT-474 cell line was selected as an optimal model for study because it represents a HER2-positive luminal B subtype breast cancer cell line. To identify the molecular mechanisms of resistance, a comprehensive transcriptomic analysis based on RNA-seq data comparison of three independent datasets including both sensitive and trastuzumab-resistant variants was applied. The methodological approach included multistep bioinformatics analysis followed by identification of regulatory interactions. The study identified genes with increased expression (FUCA2, HSPE1, SHLD1, NMD3) and genes with decreased expression (GPC5, FSTL1, ATG16L2, POLD2) in resistant cells. Key transcription factors (E2F1, MYC, YBX1, HEY1, NFIC, TFAP2A, AP-1/JUN, NCOA1) regulating the expression of the detected genes during the development of resistance were identified. The changes identified indicate a complex reprogramming of transcriptional activity affecting cell cycle processes, DNA repair, metabolism, and the epithelial-mesenchymal transition. The findings expand our understanding of the molecular mechanisms of trastuzumab resistance and open prospects for the development of novel therapeutic strategies to overcome drug resistance in HER2-positive breast cancer.