Glycerol monolaurate (GML) is a natural surfactant comprised of a monoester of glycerol and the medium-chain fatty acid lauric acid (LA). Both GML and LA have bacteriostatic properties, but less is known about the effects of these nutrients on immune or intestinal epithelial cells. The first objective of this study was to assess impacts of GML, LA, and methyl laurate (ML) on inflammatory signaling in RAW 264.7 murine macrophages transfected with a vector that drives expression of alkaline phosphatase (AP) upon activation of NFκB. RAW cells were challenged with 0.1 µg/mL LPS or not for 6 h. Medium was then replaced to provide varying doses of GML, LA and ML (0, 0.5, 2.5, 12.5, 62.5, and 312.5 µM) in 0.06% dimethyl sulfoxide (DMSO) for an additional 4 h (2 × 3 × 6 factorial, n = 6). In addition to AP activity, resazurin metabolism was used to assess cell viability. As expected, LPS treatment significantly increased AP activity and decreased cell viability, whereas DMSO mitigated the loss of viability from LPS. Therefore, the DMSO control served as the reference point for evaluating treatment effects. Increasing doses of GML, LA, and ML all decreased AP activity in a log-linear manner in the presence of LPS (P < 0.001), with significant effects detected at 62.5 and 312.5 µM for all 3 compounds (P < 0.05, Tukey test). No treatment effects were detected in the absence of LPS, and cell viability measures did not indicate toxic effects of these compounds. Second, we determined whether these compounds influence tight junction integrity in LPS-challenged intestinal cells. Caco-2 human colon cancer cells were cultured in transwell inserts for 10 d. Cells were treated with 312.5 µM GML, LA, or ML for 48 h, with or without addition of 1 μg/mL LPS for the last 24 h (n = 3). Permeability was measured by the transepithelial flux of fluorescein-sulfonic acid. LPS increased monolayer permeability, whereas all 3 compounds significantly attenuated this response (P < 0.05), with LA and GML having a greater effect than ML. The results demonstrate that LA and 2 of its esters have anti-inflammatory impacts on macrophages and reduce permeability of colonocytes challenged with LPS.