Nanodiamonds (NDs) display several attractive features rendering them useful for medical applications such as drug delivery. However, the interactions between NDs and the immune system remain poorly understood. Here, we investigated amino-, carboxyl-, and poly(ethylene glycol) (PEG)-terminated NDs with respect to primary human immune cells. We applied cytometry by time-of-flight (CyToF) to assess the impact on peripheral blood mononuclear cells at the single-cell level, and observed an expansion of plasmacytoid dendritic cells (pDCs) which are critically involved in antiviral responses. Subsequent experiments demonstrated that the NDs were actively internalized, leading to a vigorous type I interferon response involving endosomal Toll-like receptors. ND-NH and ND-COOH were more potent than ND-PEG, as evidenced by using TLR reporter cell lines. Computational studies demonstrated that NDs interacted with the ligand-binding domains of TLR7 and TLR9 with high affinity though this was less pronounced for ND-PEG. NDs with varying surface functionalities were also readily taken up by macrophages. To gain further insight, we performed RNA sequencing of a monocyte-like cell line exposed to NDs, and found that the phagosome maturation pathway was significantly affected. Indeed, evidence for lysosomal hyperacidification was obtained in dendritic cells and macrophages exposed to NDs. Moreover, using a reporter cell line, NDs were found to impinge on autophagic flux. However, NDs did not affect viability of any of the cell types studied. This study has shown that NDs subvert dendritic cells leading to an antiviral-like immune response. This has implications not only for drug delivery but also for anticancer vaccines using NDs.