NO-mediated alternative pathway plays an important role in protecting wheat seedlings against drought stress through dissipating excessive reducing equivalents generated by photosynthesis. Alternative pathway (AP) has been proven to be involved in responses to various stresses. However, the mechanisms of AP in defense response to drought stress are still lacking. The aims of this work are to investigate the role of AP in drought tolerance and how AP is induced under drought stress using two wheat cultivars with different drought tolerance. Our results showed that Longchun22 cultivar is more tolerant to drought than 98SN146 cultivar. Seedlings exposed to drought led to a significant increase in AP, and it increased more in Longchun22. Furthermore, chlorophyll fluorescence parameters (Fv/Fm, I broken vertical bar PSII, qP) decreased significantly in drought-treated seedlings, especially in 98SN146, indicating that photoinhibition occurred under drought stress. Pretreatment with SHAM, the malate-oxaloacetate shuttle activity and photosynthetic efficiency were further inhibited in drought-treated seedlings, resulting in more serious oxidative damage as indicated by higher levels of malondialdehyde and hydrogen peroxide. Moreover, NO modulated AP under drought stress by increasing AOX1a expression and pyruvate content. Taken together, these results indicate that NO-mediated AP is involved in optimizing photosynthesis under drought stress by avoiding the over-reduction of photosynthetic electron transport chain, thus reducing reactive oxygen species production and oxidative damage in wheat leaves.