Several inorganic and organic compounds including glycine betaine (GB) are presently being used as an exogenous application to enhance tolerance in plants to different environmental stresses. The current study assessed to what extent exogenously applied GB could improve the gaseous exchange capacity and primary and secondary metabolites in two accessions (16178 and 16180) of safflower (Carthamus tinctorius L.) plants under drought stress. Three-week-old plants of both safflower accessions were subjected to well-watered (control) or water-deficit conditions (60% field capacity (FC)). Three levels of GB (control, 50 mM and 100 mM) were sprayed to the foliage of the control and stressed plants after one month of drought application. After two weeks of foliar application of GB, gas exchange characteristics and other biochemical parameters were determined. The results showed that water deficiency markedly suppressed plant biomass, chlorophyll contents, photosynthesis rate (A), water use efficiency (A/E), stomatal conductance (gs) and relative water contents (RWC) of both accessions of safflower, while it enhanced the levels of osmolytes (GB and proline), hydrogen peroxide (H2O2) and total phenolics. Foliar application of GB was effective in enhancing the plant biomass, chlorophyll contents, gs, sub-stomatal CO2 concentration (Ci), Ci/Ca ratio, osmolytes, H2O2, ascorbic acid (AsA), total phenolics and RWC in safflower plants under water shortage. Thus, exogenous application of GB could be used as an effective strategy to improve plant growth, photosynthetic attributes and secondary metabolites in safflower plants under water deficit conditions.