Purpose: To explore whether probiotic supplementation could attenuate serum trimethylamine-N-oxide (TMAO) level and impact the intestinal microbiome composition. Design: Forty healthy males (20–25 years old) were randomized into the probiotic group (1.32 × 10¹¹ CFU live bacteria including strains of Lactobacillus acidophilus, Lactobacillus rhamnosus GG, Bifidobacterium animalis, and Bifidobacterium longum daily) or the control group for 4 weeks. 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. Results: 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 ± 0.97 μmol/L 8 h vs. 19.17 ± 2.55 μmol/L 8 h, P = 0.106) and the pre- to post-intervention AUC alterations (∆AUC) (− 6.33 ± 2.00 μmol/L 8 h vs. − 0.73 ± 3.04 μmol/L 8 h, 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. Conclusions: The current probiotic supplementation resulted in detectable change of intestinal microbiome composition but failed to attenuate the serum TMAO elevation after PCCT. Clinicaltrials.gov Identifier: NCT03292978. Clinicaltrials.gov website: https://clinicaltrials.gov/ct2/show/NCT03292978.