Under polyandry, males are selected to produce more competitive ejaculates. Theoretical models have explored how the mechanism of sperm competition drives males to partition investment within an ejaculate between sperm quantity and quality. The raffle-based competition model predicts that increased level of sperm competition selects for larger numbers of sperm in ejaculates. Sperm competition is also thought to promote the evolution of longer sperm, because longer sperm could be faster. In eusocial Hymenoptera, the mating system imposes unique selective pressures on male ejaculates. Males are short lived; they reach adulthood with a finite amount of spermatozoa, and they mate typically with a single or a few females and die. The actual number of spermatozoa stored in their accessory testes at emergence is thus a reliable measure of total investment into sperm production. In a comparative study of 15 species of Cataglyphis desert ants, we used phylogenetically controlled analyses to investigate relationships between levels of sperm competition, sperm production and sperm length. We measured sperm production by quantifying the number of spermatozoa present in testes, instead of using a proxy measure such as size of testes. Multiple queen mating is the ancestral state in the genus but reduction in mating frequency evolved secondarily and independently in some clades, providing a unique opportunity to examine how reduction from multiple to single mating influences sperm traits. Our results provide phylogenetically robust evidence that species experiencing greater levels of sperm competition produce more sperm. After controlling for male size, investment in sperm production decreases significantly according to the sequence obligatory multiple queen mating > multiple–single queen mating > single–double queen mating. Furthermore, the number of spermatozoa produced per male decreases significantly with reduction in paternity frequency for each species. In contrast, neither sperm length nor male size was significantly associated with the mating system classes or the number of patrilines. Our measures of sperm number provide the first direct evidence that sperm production covaries with the level of sperm competition in a eusocial insect. Given the reversal from multiple to single mating in Cataglyphis, our comparative analysis also shows convincingly that reduction in sperm competition influences sperm traits.