BACKGROUND: Rumen microorganisms are key regulators of ruminant growth and production performance. Identifying probiotic candidates through microbial culturomics presents a promising strategy for improving ruminant production performance. Our previous study identified significant differences in rumen microbial communities of Holstein calves with varying average daily gain (ADG). This study aims to identify a target strain based on the findings from multi-omics analysis and literature review, isolating and evaluating the target microbial strains from both the rumen and hindgut contents for their probiotic potential. RESULTS: Parabacteroides distasonis, a strain closely associated with ADG, was successfully isolated from calf rumen content cultured with Fastidious Anaerobe Agar (FAA) medium and named Parabacteroides distasonis F4. Whole-genome sequencing and pan-genome analysis showed that P. distasonis F4 possesses a core functional potential for carbohydrate and amino acid metabolism, with the ability to produce propionate, acetate, and lactate. The results of targeted and untargeted metabolomics further validated the organic acid production and metabolic pathways of P. distasonis F4. An in vitro simulated rumen fermentation test showed that supplementation with P. distasonis F4 significantly altered rumen microbial community structure and increased the molar proportions of propionate and butyrate in the rumen. Furthermore, an in vivo study demonstrated that dietary supplementation with P. distasonis F4 significantly increased the ADG of pre-weaning calves. CONCLUSIONS: This study represents the first isolation of P. distasonis F4 from rumen, highlighting its potential as a probiotic strain for improving rumen development and growth performance in ruminants.