Lactoferrin is extensively utilized in the food and pharmaceutical industries owing to its broad-spectrum bioactivities; however, its potential cytotoxicity to microbial hosts severely restricts the efficient construction of cell factories. To elucidate the stress response mechanisms of yeast to lactoferrin, Saccharomyces cerevisiae C800, Pichia pastoris GS115, and their corresponding recombinant strains were employed as models, and a systematic evaluation combining phenotypic detection with transcriptomic analysis was conducted. Exogenous addition assays demonstrated that bovine lactoferrin suppressed yeast growth in a dose-dependent manner. At a concentration of 0.60 g·L-1, the survival rates of the two strains were reduced to 82.58% and 78.28%, respectively, and the fluorescence values reflecting metabolic activity were decreased from 6.38×106 and 7.07×106 to 2.58×106 and 3.99×106, respectively, indicating that not only was the viable cell count reduced, but sublethal metabolic damage was also induced in surviving cells. Scanning electron microscopy revealed severe cell membrane damage in S. cerevisiae, whereas only surface roughening was observed in P. pastoris, corroborating the membrane-disrupting effects. The aforementioned inhibition was significantly antagonized by the supplementation of 10 mmol·L-1 FeCl3, with the OD600 values being restored to 138.44% and 108.76% of the control levels, respectively, suggesting that iron metabolic disturbance serves as one of the core stressors. Transcriptomic analysis revealed essential differences in the responses of the two strains. Global transcriptional repression was detected in S. cerevisiae, with 567 differentially expressed genes being identified at 8 h post-induction (81.7% downregulated). Ribosomal RNA processing genes (UTP15 and NOP15) and energy metabolism pathways were synchronously downregulated, suggesting that growth arrest was triggered to cope with the metabolic burden. In contrast, a more intense stress response was exhibited by P. pastoris, with the number of differentially expressed genes increasing to 761. The iron transporter gene FTR1 (log2FC = +2.47) and the flavohemoglobin gene YHB1 (log2FC = +1.49) were markedly upregulated, implying that disrupted iron homeostasis resulted in intracellular oxidative stress. Concurrently, chaperone genes and emergency carbon metabolism pathways were activated, reflecting extensive metabolic remodeling. In this study, the differential transcriptomic profiles of the two yeast species under lactoferrin stress were systematically characterized, providing crucial theoretical references for the targeted optimization of yeast chassis to enhance lactoferrin production.