Salt-inducible kinase pan-inhibitors suppress production of TLR ligand-induced pro-inflammatory cytokines in myeloid cells and potentiate production of IL-10. To define the role of specific SIK isoforms (SIK1, SIK2, and SIK3) in induced cytokine production, we created SIK isoform single knockout (KO) THP-1 cell lines. SIK isoform expression in THP-1s is similar to that of primary human macrophages and dendritic cells with SIK2 and SIK3 expressed ~10-fold higher than SIK1. Upon TLR ligand stimulation SIK3 KO cells displayed a significant reduction in TNFα production, with SIK2 KO cells having a modest reduction, and SIK1 KO cells having no reduction in TNFα compared to controls. SIK2/1 or SIK3/1 double KOs showed similar TNFα production to their parental SIK2 or SIK3 single KO, respectively, confirming that SIK1 is dispensable. SIK2/3 double KO cells, however, lead to a significant reduction in TNFα production compared to their parental single KO lines indicating some overlapping function. In addition, we observed that SIK3-deficient THP-1 cells proliferated more slowly than SIK1 or SIK2 KO clones and that SIK2/3 double KOs led to a complete arrest of cell proliferation and induction of a macrophage-like morphology after 12-days in culture. We conclude that SIK3 and SIK2 are critical regulators of TLR-induced TNFα production and proliferation in THP-1 cells, with SIK3 having a more dominant role than SIK2 and no apparent role for SIK1.