To explore whether probiotic supplementation could attenuate serum trimethylamine-N-oxide (TMAO) level and impact the intestinal microbiome composition. Forty healthy males (20-25years old) were randomized into the probiotic group (1.32 × 1011CFU live bacteria including strains of Lactobacillus acidophilus, Lactobacillus rhamnosus GG, Bifidobacterium animalis, and Bifidobacterium longum daily) or the control group for 4weeks. All participants underwent a phosphatidylcholine challenge test (PCCT) before and after the intervention. Serum TMAO and its precursors (TMA, choline and betaine) were measured by UPLC-MS/MS. The faecal microbiome was analyzed by 16S rRNA sequencing. Serum TMAO and its precursors were markedly increased after the PCCT. No statistical differences were observed in the probiotic and the control group in area under the curve (AUC) (14.79 ± 0.97μmol/L8h vs. 19.17 ± 2.55μmol/L8h, P = 0.106) and the pre- to post-intervention AUC alterations (∆AUC) (-6.33 ± 2.00μmol/L8h vs. -0.73 ± 3.04μmol/L8h, P = 0.131) of TMAO; however, higher proportion of participants in probiotic group showed their TMAO decrease after the intervention (78.9% vs. 45.0%, P = 0.029). The abundance of Faecalibacterium prausnitzii (P = 0.043) and Prevotella (P = 0.001) in the probiotic group was significantly increased after the intervention but without obvious differences in α- and β-diversity. The current probiotic supplementation resulted in detectable change of intestinal microbiome composition but failed to attenuate the serum TMAO elevation after PCCT. CLINICALTRIALS. NCT03292978. CLINICALTRIALS.GOV WEBSITE: https://clinicaltrials.gov/ct2/show/NCT03292978 .