Atopic dermatitis is a chronic condition where epidermal barrier dysfunction and cytokine production by infiltrating immune cells exacerbate skin inflammation and damage. A total lipid extract from Macrocystis pyrifera, a brown seaweed, was previously reported to suppress inflammatory responses in monocytes. Here, treatment of human HaCaT keratinocytes with M. pyrifera lipids inhibited tumour necrosis factor (TNF)-α induced TNF receptor-associated factor 2 and monocyte chemoattractant protein (MCP)-1 protein production. HaCaT cells stimulated with TNF-α, interleukin (IL)-4, and IL-13 showed loss of claudin-1 tight junctions, but little improvement was observed following lipid pre-treatment. Three-dimensional cultures of HaCaT cells differentiated at the air–liquid interface showed increased MCP-1 production, loss of claudin-1 tight junctions, and trans-epidermal leakage with TNF-α, IL-4, and IL-13 stimulation, with all parameters reduced by lipid pre-treatment. These findings suggest that M. pyrifera lipids have anti-inflammatory and barrier-protective effects on keratinocytes, which may be beneficial for the treatment of atopic dermatitis or other skin conditions.