Gas exchange, chlorophyll fluorescence, and contents of some metabolites in two Japanese honeysuckle (Lonicera japonica Thunb.) cultivars, Damaohua (2n = 2x) and Jiufengyihao (2n = 4x), were compared with explore the function of chromosome doubling under water stress conditions. Water stress significantly decreased net photosynthesis rate, stomatal conductance, and transpiration rate of both cultivars. It also decreased electron transport rate, effective quantum yield of Photosystem II, photochemical quenching, and starch content, but increased non-photochemical quenching and contents of total soluble sugars, proline, and malondialdehyde. However, the tetraploid cultivar showed higher resistance to water stress than the diploid, as indicated by the fact that gas exchange, chlorophyll fluorescence, and metabolites were less affected for the tetraploid than the diploid. Moreover, the tetraploid recovered more quickly than the diploid after re-watering. Morphological and anatomical analysis further revealed that the tetraploid possessed less whole plant leaf area, higher leaf mass per unit area, thicker epidermis (both upper and lower) and palisade tissue, as well as denser pubescence. All of those specialised structures caused by chromosome doubling might lead to greater capacity in coping with drought stress. Our findings suggest that the effect of chromosome doubling on drought resistance in L. japonica could attribute to the improvement of structure and photosynthesis-related traits.