Calanus sinicus is an important copepod in shelf waters of the western North Pacific. The objective of this study was to investigate the occurrence of eukaryotes and prokaryotes in the natural diet of adults and C5 copepodites of C. sinicus. Hypervariable regions of the eukaryotic 18S and prokaryotic 16S rRNA genes were amplified from the gut contents of C. sinicus collected from coastal seas around Taiwan and Hong Kong and sequenced by Illumina sequencing. Results showed that the diets of adults and C5 copepodites of C. sinicus were similar. However, the diet of C. sinicus seems to be different between the two sampling locations. Diatoms were detected in C. sinicus samples collected from both areas, whereas dinoflagellates were only detected in samples from Hong Kong. Hydrozoans were found to be common in C. sinicus samples from Taiwan, whereas ctenophores were common in samples from Hong Kong. The cyanobacterium Synechococcus was recovered in samples from both areas. Our results have provided DNA-based evidence to support previous suggestions that C. sinicus could feed omnivorously on a diverse assemblage of phytoplankton, animals and bacteria. The ability of C. sinicus to feed on the small larvae of their predators such as ctenophores may have important implications on future analyses of the marine planktonic food web. •DNA-based technique was used to study the natural diet of Calanus sinicus.•C. sinicus feeds omnivorously on a diverse assemblage of eukaryotes and prokaryotes.•C. sinicus can feed on the juveniles of their potential predators.•Our findings have important implications on future analyses of the marine planktonic food web.