Honeybees cannot synthesize arachidonic acid (ARA) themselves, only obtain it from food. Most pollen is deficient or contains a small amount of ARA. The necessity of supplementary ARA in bees’ diet has not been studied. The objective of this study was to investigate the effects of dietary ARA levels on the growth and immunity of Apis mellifera ligustica. A total of 25 honeybee colonies were randomly assigned to five dietary groups which were fed basic diets supplemented with 0, 2, 4, 6, and 8% of ARA. The diet with 4% ARA improved the body weight of newly emerged worker bees compared with the control group. Supplement of ARA in honeybee diets changed the fatty acid composition of honeybee body. SFA and MUFA contents of bees’ body declined, and PUFA content rised in the ARA group. Compared with the control group, the supplement of ARA in honeybee diets increased the contents of ARA, C22:6n-3 (DHA) and C18:3n-6 in bees’ body significantly, but decreased the contents of C16:1 and C18:3n-3. The diet supplied with 4% ARA reduced the mortality rate of honeybee infected with Escherichia coli. The activity of immune enzymes (phenoloxidase, antitrypsin, and lysozyme) and the mRNA expression levels of immune genes (defensin-2, toll, myd88, and dorsal) were improved by ARA diets to varying degrees depending on the ARA levels, especially 4% ARA. These results suggested that dietary ARA could improve the growth, survival, and immune functions of honeybees. Supplement of ARA in bees’ diet would be valuable for the fitness of honeybees.