Background and aims <p>Glyphosate is a common herbicide in peach orchards, but its long-term impacts on tree physiology and soil health remain unclear. This study aims to comprehensively evaluate the effects of long-term glyphosate application on the growth of peach trees, soil properties, and microbial communities.</p> Methods <p>In this study, through a six-year continuous positioning experiment, combined with the analysis of physiological and biochemical indexes and high-throughput sequencing technology, the multi-dimensional impacts of glyphosate on the peach tree-soil system were systematically revealed.</p> Results <p>The results showed that compared with the control group, the glyphosate treatment group significantly inhibited the photosynthetic function of peach trees. The net photosynthetic rate, stomatal conductance, and water use efficiency decreased synchronously, accompanied by an imbalance in the antioxidant system. The glyphosate-derived metabolite aminomethylphosphonic acid (AMPA) accumulated in soil at concentrations markedly exceeding those of the parent compound, inducing soil acidification, organic matter loss, and diminished available potassium, while simultaneously stimulating urease activity. The diversity and structure of the soil microbial community changed significantly. Shifts in the abundance of dominant bacterial communities, such as <i>Proteobacteria</i>, showed significant correlations with soil nutrients and enzyme activities.</p> Conclusions <p>Long-term glyphosate application negatively affects peach tree physiology and soil ecological function. These findings provide critical insights for optimizing herbicide use and soil management practices in peach orchards.</p> Graphical Abstract <p></p>