The cyclopoid copepod Apocyclops royi (Lindberg 1940) is one of two dominant mesozooplankton species in brackish Taiwanese aquaculture ponds. Periodically low n-3 polyunsaturated fatty acid (PUFA) content in seston could potentially be a limiting factor for zooplankton diversity. Apocyclops royi’s potential ability to biosynthesize n-3 PUFA was investigated through a short-term feeding experiment on four species of microalgae. Furthermore, we analyzed the expression of genes encoding putative fatty acid elongase (ELO) and desaturase (FAD) enzymes in A. royi on long-term diets of the PUFA-poor Dunaliella tertiolecta and the PUFA-rich Isochrysis galbana. The copepods exhibited high contents of docosahexaenoic acid (DHA, C22:6n-3) (>20% of total fatty acid) even when DHA-starved for two generations, and no significant differences were found in absolute DHA content between treatments. Transcripts correlating to the four enzymes Elovl4, Elovl5, Fad Δ5 and Fad Δ6 in the n-3 PUFA biosynthetic pathway were identified. Gene expression analysis revealed a significantly higher expression of two desaturases similar to Fad Δ6 in copepods fed PUFA-lacking algae compared to copepods fed algae with high PUFA content. These findings suggest a highly active n-3 PUFA biosynthesis and capability of DHA production in A. royi when fed low-PUFA diets.