To understand the association between the cytokine network and psoriasis, the present study cultured human keratinocytes (HaCaT cells) and investigated the effects of the phosphatidylinositol-3-kinase (PI3K) p55 regulatory subunit (p55PIK), and its N-terminal 24 amino acids (N24) on the regulation of endotoxin (LPS)-induced cytokine secretion. The results of the enzyme-linked immunosorbent assay and reverse transcription quantitative polymerase chain reaction revealed an increased release of the inflammatory cytokines, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-8 in the HaCaT cells following LPS stimulation. Transfection with the adenovirus (AD)-N24-green fluorescent protein (GFP) suppressed the release of these cytokines, whereas AD-p55PIK-GFP increased their release. Immunocytochemistry detected a low level of nuclear factor (NF)-kappa B p65 staining in quiescent HaCaT cells, which was localized primarily in the cytoplasm. LPS stimulation induced the translocation of NF-kappa B p65 protein into the nucleus and intense staining suggested increased expression. Transfection with AD-N24-GFP reduced the expression of NF-kappa B p65 in the nucleus. Western blot analysis demonstrated that AD-N24-GFP downregulated the expression levels of the Toll-like receptor (TLR)2/TLR4/myeloid differentiation factor 88 (MyD88) pathway components in the HaCaT cells, without affecting the PI3K/Akt signaling pathway. Transfection with AD-p55PIK-GFP resulted in an increased expression level of MyD88 protein and phosphorylated Akt. Co-transfection with AD-N24-GFP and AD-p55PIK-GFP did not significantly alter the levels of phosphorylated extracellular-signal-regulated kinases 1/2, c-Jun N-terminal kinases or p38, indicating that AD-N24-GFP and AD-p55PIK-GFP did not affect the mitogen-activated protein kinase signaling pathway. In conclusion, AD-N24-GFP effectively inhibited the LPS-induced expression levels of TNF-alpha, IL-6 and IL-8. The elevated expression of p55PIK synergized with LPS and promoted the release of inflammatory cytokines. AD-N24-GFP and AD-p55PIK-GFP affected LPS-induced inflammatory cytokine release in the HaCaT cells through the TLRs/MyD88 signaling pathways.