Exposure to fibrogenic multi-walled carbon nanotubes (MWCNTs) induces the production of proinflammatory lipid mediators (LMs) in myeloid cells to instigate inflammation. The molecular underpinnings of LM production in nanotoxicity remain unclear. Here we report that PU.1, an ETS domain-containing master regulator of hematopoiesis, critically regulates the induction of arachidonate 5-lypoxygenase (Alox5) and the production of LMs. MWCNTs (Mitsui-7) at 2.5 or 10 µg/mL induced the expression of Alox5 in murine and human macrophages at both mRNA and protein levels, accompanied by marked elevation of chemotactic LM leukotriene B4 (LTB4). Induction is comparable to those by potent M1 inducers. Carbon black, an amorphous carbon material control, did not increase Alox5 expression or LTB4 production at equivalent doses. MWCNTs induced the expression of a heterologous luciferase reporter under the control of the murine Alox5 promoter. Deletional analysis of the 2 kb promoter uncovered multiple inhibitory and activating activities. The proximal 250 bp region had the largest activation that was further increased by MWCNTs. The Alox5 promoter contains four PU box-like enhancers. PU.1 bond to each of the enhancers constitutively, which was further increased by MWCNTs. Knockdown of PU.1 using specific small hairpin-RNA blocked the basal and induced expression of Alox5 and the production of LTB4 as well as prostaglandin E2. The results demonstrate a critical role of PU.1 in mediating MWCNTs-induced expression of Alox5 and production of proinflammatory LMs, revealing a molecular framework where the hematopoietic transcription factor PU.1 is activated to orchestrate multiple proinflammatory responses to sterile particulates.