In vivo studies have suggested the immunomodulatory properties of Septilin, an herbal preparation. This drug is being used against various types of inflammatory disorders. However, the mechanism of action of Septilin in the modulation of inflammation is not explored using suitable in vitro models. Hence, we decided to study the modulatory role of Septilin in lipopolysaccharide (LPS) mediated signaling in macrophage and monocyte cells. It was observed from the present study that by employing tumor necrosis factor α (TNF-α) bioassay and reverse transcription-polymerase chain reaction (RT-PCR), Septilin inhibited TNF-α production in LPS (1 μg/mL) stimulated RAW 264.7 cells (p < 0.05). 80% inhibition of TNF-α was observed even at 2.5% Septilin. Septilin at all the concentrations tested could also significantly block the LPS mediated nitric oxide (NO) production (p < 0.01) and expression of inducible NO synthase (iNOS) gene. LPS mediated interleukin 6 (IL-6) and IL-8 production was also blocked by Septilin at the concentrations tested. This herbal preparation could also inhibit cycloxygenase 2 (COX-2) activity and suppression of COX-2 and phosphodiesterase 4 B (PDE4B) mRNA expression in a concentration dependent manner. Taken together, these findings from the present in vitro study suggest the anti-inflammatory and immunomodulatory properties of Septilin.