In inflammatory diseases, tissue damage is critically associated with nitric oxide (•NO) and cytokines which are overproduced in response to cellular release of endotoxins. Here we investigated the inhibitory effect of roscovitine, a selective inhibitor of cyclin-dependent kinases (CDKs) on •NO production in mouse macrophages. In RAW264.7 cells we found that roscovitine abolished the production of •NO induced by lipopolysaccharide (LPS). Moreover, roscovitine significantly inhibited LPS-induced inducible nitric oxidase synthase (iNOS) mRNA and protein expression. Our data also showed that roscovitine attenuated LPS-induced phosphorylation of IKK, IB and p65 but enhanced the phosphorylation of ERK, p38 and JNK. In addition, roscovitine dose-dependently inhibited LPS-induced expression of COX-2, IL-1 and IL-6 but not TNF. Tetrahydrobiopterin (BH4), an essential cofactor for iNOS, is easily oxidized to 7, 8-dihydrobiopterin (BH2). Roscovitine significantly inhibited LPS-induced BH4 biosynthesis and decreased BH4/BH2 ratio. Furthermore, roscovitine greatly reduced the upregulation of GTP cyclohydrolase 1 (GCH-1), the rate-limiting enzyme for BH4 biosynthesis. Using other CDK inhibitors, we found that CDK1, CDK5, and CDK7, but not CDK2, significantly inhibited LPS-induced •NO production in macrophages. Similarly, in isolated peritoneal macrophages, roscovitine strongly inhibited •NO production, iNOS and COX-2 upregulation, activation of NFB and induction of GCH-1 by LPS. Together, our data indicates that roscovitine abolishes LPS-induced •NO production in macrophages by suppressing NFB activation and BH4 biosynthesis, which might be mediated by CDK1, CDK5 and CDK7. Our results also suggest that roscovitine may inhibit inflammation and that CDKs may play important roles in the mechanisms by which roscovitine attenuates inflammation.