Anopheline mosquitoes are major vectors of malaria parasites. When the gametocytes of the malaria parasite are transferred from a vertebrate to mosquitoes, they differentiate into gametes, and are fertilized in the midguts of mosquitoes. Xanthurenic acid (XA), a waste product of the ommochrome synthesis pathway, has been shown to induce exflagellation during microgametogenesis in vitro; however, it currently remains unclear whether endogenous XA affects the infectivity of anopheline mosquitoes to malaria parasites in vivo due to the lack of appropriate experimental systems such as a XA-deficient line. In the present study, we produced a XA-deficient line in Anopheles stephensi using transcription activator-like effector nuclease (TALEN)-mediated gene targeting (knockout) of the kynurenine 3-monooxygenase (kmo) gene, which encodes an enzyme that participates in the ommochrome synthesis pathway. The knockout of kmo resulted in the absence of XA, and oocyst formation was inhibited in the midguts of these XA-deficient mosquitoes, which, in turn, reduced sporozoite numbers in their salivary glands. These results suggest that endogenous XA stimulates exflagellation, and enhances the infectivity of anopheline mosquitoes to malaria parasites in vivo. The XA-deficient line of the anopheline mosquito provides a useful system for analyzing and understanding the associated factors of malaria gametogenesis in the mosquito midgut.