Abstract Purpose To determine serum fatty acids of lacto-ovo vegetarian (LOV), vegan (VEG) and omnivorous (OMN) adults, and to analyse the effects of consuming a docosahexaenoic acid (DHA) supplement of vegetable origin on fatty acid profile. Methods A randomized, double-blind, placebo-controlled crossover design was conducted in healthy adults. Volunteers (n = 116) were randomly assigned to a DHA-supplement (dose 250 mg/day), made from the microalgae Schizochytrium sp., or a placebo during 5-week periods separated by a 5-week washout interim period. Compliance and dietary intake were estimated and serum fatty acids were determined by gas chromatography. Results were analysed by mixed linear models. Results Percentage of linoleic acid (C18:2n6) in serum was the highest among the fatty acids in the three diet groups, followed by oleic (C18:1n9) and palmitic (C16:0) acids. Linoleic (C18:2n6) and alpha-linolenic (C18:3n3) acids were higher in VEG compared to OMN (p < 0.001), while in LOV, their levels were intermediate between the other groups. Women presented higher DHA (C22:6n3) than men (p < 0.001). The DHA-supplement increased serum DHA compared to placebo in the three diet groups (p < 0.001), and a higher increase was observed in VEG followed by LOV (p < 0.001). The ratio serum n-6/n-3 improved by the supplementation but remained higher in LOV and VEG than in OMN. In contrast, the DHA-supplement decreased docosapentaenoic (C22:5n3) and docosatetraenoic (C22:4n6) acids in all diet groups (p < 0.001) and increased the eicosapentaenoic to alpha-linolenic fatty acids ratio (p = 0.016). Conclusion The DHA-supplement at dose of 250 mg/day was effective in increasing serum DHA either in omnivorous, lacto-ovo vegetarian and vegan adults. Clinical trial registration Registered at Clinicaltrials.gov (www.clinicaltrials.gov), NCT04278482.