The study aims to investigate the impacts of hyperglycemia in the presence of fatty acids on early placentation processes that involves tube formation, cellular growth and proliferation, and metabolic activities of the first trimester trophoblast cells. Effects of maternal circulatory glucose levels that mimic physiological (5.5 mM), pre-diabetic (11 mM) and diabetic (≥25 mM) phenotypes on tube formation (as a measure of angiogenesis in vitro), cellular viability and proliferation, fatty acid uptake and expression of genes associated with invasion, angiogenesis and fatty acid metabolism were examined using HTR8/SVneo cells. Glucose (25 mM) induced tube formation, viability, and proliferation of the first trimester trophoblast cells, HTR8/SVneo. Tube formation was, however, disintegrated in the presence of high glucose (40 mM) which was partially protected by eicosapentaenoic acid, 20:5n-3 (EPA) and docosahexaenoic acid, 22:6n-3 in vitro. Glucose (25 mM)-mediated induction in tube formation was favored by increased cellular uptake of [(14)C]EPA (p < 0.05). Treatment of HTR8/SVneo cells with glucose (25 mM) significantly increased mRNA and protein level of matrix metalloproteinase-9 (MMP9) (p < 0.05). In addition, glucose (25 mM) stimulated the expression of fatty acid binding protein-4, FABP4, and plasma membrane fatty acid binding protein, FABPpm, in these cells (p < 0.05). Glucose-stimulated tube formation in a 'concentration-dependent' manner, and this may involve activation of several factors that include MMP9 and fatty acid uptake and metabolism.