Climate change is causing the spread of common ragweed across Europe, along with rising ground-level ozone (O3) in semi-urban and rural environments. Although elevated O3 has been shown to affect pollen-intrinsic allergenic compounds, here we addressed the lack of in vivo evidence on how ozone-exposed ragweed pollen modulates allergic responses. Ragweed plants were grown in controlled plant growth chambers under control (40 ppb) or elevated (80 ppb and 120 ppb) O3 levels. Aqueous pollen extracts (RWE) from control- or O3-exposed plants were administered in vivo in a murine model for allergic airway inflammation (AAI) and employed in a human in vitro system of monocyte-derived dendritic cells (DCs), key initiators of the allergic response. Adjuvant factors and metabolites in control- and O3-RWE were investigated using ELISA and untargeted metabolomics. Compared to control-RWE, 80 ppb O3 induced a statistically significant enhancement of few AAI parameters, whereas 120 ppb O3 yielded statistically significant dampening effects on AAI. On the same line, in human DCs isolated from atopic donors, RWE O3 80 ppb slightly increased, while RWE O3 120 ppb decreased the expression of maturation markers. Metabolomic profiling revealed pronounced, dose-dependent shifts in pollen primary and secondary metabolites, with moderate stimulation of pro-inflammatory lipid- and protein-derived mediators at 80 ppb, whereas higher O3 levels (120 ppb) induced metabolic degradation resulting in reduced lipid and protein pro-allergenic compounds. Overall, ozone altered ragweed pollen allergenicity in a dose-dependent manner through plant physiological responses to oxidative stress, highlighting the interaction between air pollution and plant physiology in shaping pollen allergenicity in a changing climate.