<p>This study investigated the herbicidal effects of glyphosate on the invasive perennial weed <i>Chromolaena odorata.</i> Natural weed populations of selected 1.2&#xa0;m in height plants were sprayed with 1% glyphosate. Measurements of gas exchange, chlorophyll fluorescence and glyphosate concentrations were taken five consecutive days after spraying in late summer (February 2023). Glyphosate was determined by high-performance liquid chromatography (HPLC). After herbicide application, CO₂ uptake decreased from 9.5 µmol m^− 2s^− 1 on day 1 to 0.95 µmol m^− 2s^− 1 on day 5. Leaf conductance and transpiration followed trends similar to those of CO₂ uptake. Saturation of CO₂ uptake occurred at a Photosynthetic Photon Flux Density (PPFD) of 1750 µmol m^− 2s^− 1 and at a leaf conductance of 0.09&#xa0;mol m^− 2s^− 1 on day one. Trends in photosynthetic quantum yield (Φ_PSII), electron transport rate (ETR) through Photosystem II (PSII) and maximum quantum yield (F_v/F_m) were similar to those of CO₂ uptake. There was a linear relationship between CO₂ uptake and leaf conductance, CO₂ uptake and transpiration and CO₂ uptake and F_v/F_m over all days. Initially, glyphosate concentrations were high in leaves and stems but declined with time. After day three, most glyphosate was located in roots, indicating transport from above to below-ground organs. Significant decreases in CO₂ uptake, Φ_PSII, and ETR through PSII and F_v/F_m indicate the inhibition of the shikimate pathway by glyphosate. The responses suggests that glyphosate was easily absorbed and translocated throughout the plant, resulting in wilting, abscission and 100% mortality within six days. This work provides novel information on the ecophysiological mechanism of glyphosate action in controlling <i>C. odorata.</i></p>