Alpha-linolenic acid (ALA), which is an n-3 polyunsaturated fatty acid (PUFA), influences honey bee feed intake and longevity. The objective of this study was to research the effect of six dietary ALA levels on the growth and development of Apis mellifera ligustica colonies. In the early spring, a total of 36 honey bee colonies of equal size and queen quality were randomly allocated into 6 groups. The six groups of honey bees were fed a basal diet with supplementation of ALA levels at 0 (group A), 2 (group B), 4 (group C), 6 (group D), 8 (group E), and 10% (group F). In this study, there were significant effects of pollen substitute ALA levels on the feeding amounts of the bee colony, colony population, sealed brood amount, and weight of newly emerged workers (P<0.05). The workers’ midgut Lipase (LPS) activity of group C was significantly lower than that of the other groups (P<0.01). The worker bees in groups B, C, and D had significantly longer lifespans than those in the other groups (P<0.05). However, when the diets had ALA concentrations of more than 6%, the mortality of the honey bees increased (P<0.01). These results indicate that ALA levels of 2 ~ 4% of the pollen substitute were optimal for maintaining the highest reproductive performance and the digestion and absorption of fatty acids in honey bees during the period of spring multiplication. Additionally, ALA levels of 2 ~ 6% of the pollen substitute, improved worker bee longevity.