Anopheline mosquitoes are the sole vectors of malaria and rely on olfactory cues for host seeking in which ammonia derived from human sweat plays an essential role. To investigate the function of the Anopheles coluzzii ammonium transporter (AcAmt) in the mosquito olfactory system, we generated an AcAmt null mutant line using CRISPR/Cas9. AcAmt-/- mutants displayed a series of novel phenotypes compared with wild-type mosquitoes including significantly lower insemination rates during mating and increased mortality during eclosion. Furthermore, AcAmt-/- males showed significantly lower sugar consumption while AcAmt-/- females and pupae displayed significantly higher ammonia levels than their wild-type counterparts. Surprisingly, in contrast to previous studies in Drosophila that revealed that the mutation of the ammonium transporter (DmAmt) induces a dramatic reduction of ammonia responses in antennal coeloconic sensilla, no significant differences were observed across a range of peripheral sensory neuron responses to ammonia and other odorants between wild-type and AcAmt-/- females. Taken together, these data support the existence of a unique ammonia-sensing mechanism in mosquitoes and that the ammonium transporter may be an important molecular target for vector control.