Biochemical properties of polyunsaturated fatty acids (PUFAs) are fundamental to sperm movements. Amongst all adjustments operated during epididymal maturation, sperm membrane lipid composition is remodelled. Specifically, the proportion of PUFAs usually increases from the caput towards the cauda epididymidis. In mammals, PUFAs are predominantly acquired through the diet, which can consequently impact male fertility. We aimed at analysing to what extent n-6 and n-3 PUFAs are incorporated into sperm in the Seba’s short-tailed bat (Carollia perspicillata), and at demonstrating the effect of the sperm fatty acid composition on sperm mobility. We therefore provided food varying in fatty acid composition to males of C. perspicillata and measured the fatty acid composition and mobility traits in spermatozoa collected from the caput and cauda epididymides. We found that n-6 and n-3 PUFAs and saturated fatty acids were significantly related to sperm velocity but not to the proportion of progressive sperm (i.e. motility). Concomitant to an increase in sperm velocity, the level of fatty acid saturation increased from the caput to the cauda epididymidis, while the proportion of PUFAs remained similar along the epididymis. A reduction in n-6 PUFAs counterbalanced an increase in n-3 PUFAs. The food treatments did not affect the sperm fatty acid composition. Our results suggest that a precise endogenous control rather than dietary effects determines sperm fatty acid composition in C. perspicillata.