The activity of kinase suppressor of ras (KSR), a kinase or a molecular scaffold upstream from Raf‐1, is involved in the MEK/ERK MAP kinase cascade which can signal cell growth, survival, or differentiation, depending on the cellular context. We provide evidence here that KSR is upregulated in HL60 cells undergoing differentiation induced by low (0.3–3 nM) concentrations of 1,25‐dihydroxyvitamin D₃ (1,25D₃), and an antisense oligo (AS), but not a sense oligo, to KSR inhibits this differentiation. The inhibition of differentiation by AS–KSR oligo was less apparent when the concentration of 1,25D₃ was increased, suggesting that at the higher concentrations of 1,25D₃ KSR is not essential for the signaling of the differentiated phenotype. The reduced differentiation of HL60 cells exposed to AS–KSR was paralleled by reduced phosphorylation of Raf‐1 Ser 259, and of p90RSK, used here as read‐out for MAPK cascade activity. Conversely, ectopic expression of Flag‐tagged wild type KSR potentiated the differentiation‐inducing effects of low concentrations of 1,25D₃. Additional data suggest that the kinase activity of KSR is required for these effects, as transfection of a kinase inactive KSR construct did not significantly increase the 1,25D₃‐induced differentiation. Enzyme assays performed with KSR immunoprecipitated from 1,25D₃‐treated cells showed kinase activity when recombinant Raf‐1 was used as the substrate, but not when the 1,25D₃‐treated cells were pretreated with AS–KSR oligos. Taken together, these data suggest that KSR participates in signaling of monocytic differentiation by augmenting the strength of the signal transmitted through Raf‐1 to downstream targets. J. Cell. Physiol. 198: 333–342, 2004© 2003 Wiley‐Liss, Inc.