Multiwalled carbon nanotubes (MWCNTs) offer immense opportunities to deliver drug and biomolecules to targeted tissues. However, it’s unclear for us about their effects on fat metabolism. Here, we demonstrate that nitrogen-doped carboxylate-functionalized MWCNTs (N-MWCNTs) inhibit fat deposition both in vivo and in vitro primarily by suppressing adipogenesis. N-MWCNTs show good biocompatability in HEK293 mammalian cells. Intramuscular administration of N-MWCNTs does not affect the body weight gain and feed intake of mice, but reduces the fat mass. In in vitro-cultured adipocytes, N-MWCNTs suppress fat accumulation, accompanying with decreased and increased expression of adipogenic and lipolysis genes, respectively. Transcriptome analysis further certified the N-MWCNT alteration of fat metabolism-related genes. Interestingly, we observed the phagocytosis of N-MWCNTs by macrophage-like cells via TEM imaging. The mRNA sequencing data also showed remarkable variation of the genes involved in TLRs pathway, ultimately leading to down- or up-regulation of inflammatory factors, of which Tnfα, Il1, Il7, Il10, and Il12 are decreased, whereas Il6 and Il11 are increased. In conclusion, N-MWCNTs induce the production of inflammatory cytokines through immune responses, which trigger the reduction of fat deposition. These findings support the usage of N-MWCNTs as a promising delivery for anti-obesity agents.