We investigated the effect of diel variation in cell properties of picoplankton prey and the intrinsic grazing rhythm of heterotrophic nanoflagellates (HNF) on the diel periodicity of HNF grazing. To achieve this aim, 2-point dilution experiments with short incubation time were performed over 24 h in the South China Sea and Hong Kong (HK) coastal water to assess the prey cell properties and HNF grazing. Fluorescently labeled bacteria (FLB) were added for inference of the intrinsic diel rhythm of HNF feeding. Cell sizes of in situ prey at both study sites and the carbon to nitrogen molar ratio (C:N) of picoplankton prey in HK coastal water exhibited an increasing trend during the day, indicating cellular growth of prey during the period. Higher grazing rates on FLB during the day compared with night at both study sites suggested higher intrinsic HNF feeding during the day. Diel patterns of HNF grazing with increasing trend during the day and decreasing trend at night were observed for all in situ prey at both study sites. Statistical analysis suggests that both time of day and prey C:N could have significant effects on the diel variation of HNF feeding.