While Kruppel-like factors (KLFs) are known to regulate many basic processes of macrophages, their modulatory roles in nanomaterial (NM)-caused bio-effects in macrophages are unclear. Recently we reported diameter-dependent induction of cytotoxicity, inflammation and lipid accumulation by multi-walled carbon nanotubes (MWCNTs) in THP-1 macrophages in vitro, and hereby we further investigated the roles of KLFs. RNA-sequencing data showed that MWCNTs diameter-dependently up-regulated a number of KLFs, including KLF6, a transcription factor (TF) mediating inflammatory macrophage polarization. Furthermore, MWCNTs influenced KLF6-related gene ontology (GO) terms and downstream genes (identified by ingenuity pathway analysis), typically those related with inflammation and TF functions. We further verified that only MWCNTs of the smallest diameter promoted soluble monocyte chemotactic protein 1 (sMCP-1) proteins, while KLF6 siRNA remarkably inhibited C–C motif chemokine ligand 2 (CCL2, MCP-1 encoding gene) mRNA. Similarly, the TF involved in endoplasmic reticulum (ER) stress, activating transcription factor 3 (ATF3), was activated by MWCNTs with the smallest diameter and inhibited by KLF6 siRNA. However, KLF6 knockdown did not obviously alter lipid levels in MWCNT-exposed macrophages. In mice receiving repeated intratracheal instillation of MWCNTs (128 μg per mouse, once a week for 5 weeks), MWCNTs of the smallest diameter induced inflammatory responses in lungs and promoted the expression of KLF6, CCL2, ATF3 and nitric oxide synthase 2 (nos2) in bronchoalveolar lavage cells. Our results suggested a previously unknown role of KLF6 in regulating MWCNT-induced inflammatory responses both in vitro and in vivo.