Pseudostellaria heterophylla (Miq.) Pax (P. heterophylla) was a valued traditional Chinese herbal medicine. Previous studies have shown that P. heterophylla TuMV spreads during the vegetative propagation cycle using tuberous roots as carriers. However, the transmission mechanism of TuMV in P. heterophylla and its effects on host growth remain to be elucidated. In this study, virus-free P. heterophylla culture seedlings were infected with control, TuMV-ZR, and TuMV-ZR-EGFP, thereby resulting in the initial infection cycle of IF1 (TIF1, TEIF1) and control NIF1, and used these roots to propagate the subsequent infection cycle IF2 (TIF2, TEIF2) and control NIF2. The transmission of TuMV-ZR seedlings was tracked by EGFP signal, and their yield, quality, and resistance were analyzed simultaneously in the critical growth period of the plants. The results indicated that TuMV-ZR accumulated in the tuberous roots of IF1 plants, subsequently migrated to IF2 during seedling growth, and was re-stored in IF2 tuberous roots, thereby forming a simple virus transmission cycle. Meanwhile, the tuberous roots of IF1 and IF2 P. heterophylla showed lower fresh weight, dry weight, soluble sugar, and saponin levels compared to NIF1 and NIF2, respectively. TuMV caused a significant reduction in chlorophyll synthesis in IF1 and IF2 P. heterophylla, resulting in impairment to their photosynthetic organs and efficiency. The measurement of stress resistance in IF1 and IF2 P. heterophylla revealed that continuous viral infection disrupted antioxidant enzyme activity, increased the content of MDA, enhanced the activity of PAL, and elevated the levels of intracellular osmotic substances in both propagation cycles. The findings indicated that the accumulation of the TuMV-ZR virus during two successive vegetative propagation cycles induced physiological stress, impaired photosynthesis, and caused progressive yield and quality decline with each cycle. This study systematically examined the impact of TuMV-ZR persistence during vegetative propagation on key physiological and biochemical indices in P. heterophylla, providing critical data to clarify vegetative-propagation-mediated germplasm degradation.