This study evaluated the effects of dietary concentrate levels and 2‐methylbutyrate (2
MB) supplementation on performance, ruminal fermentation, bacteria abundance, microbial enzyme activity and urinary excretion of purine derivatives ( PD) in steers. Eight ruminally cannulated Simmental steers (12 months of age; 389 ± 3.7 kg of body weight) were used in a replicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement. Moderate‐concentrate (400 g/kg diet [ MC]) or high‐concentrate (600 g/kg diet [ HC]) diets were fed with or without 2 MB(0 g/day [2 MB−] or 15.0 g/day [2 MB+]). Dry matter intake and average daily gain increased, but feed conversion ratio decreased with the HCdiet or 2 MBsupplementation. Ruminal pHdecreased, but total volatile fatty acid increased with the HCdiet or 2 MBsupplementation. Molar proportion of acetate and acetate‐to‐propionate ratio decreased with the HCdiet, but increased with 2 MBsupplementation. Propionate molar proportion and ruminal NH3‐N content increased with the HCdiet, but decreased with 2 MBsupplementation. Neutral detergent fibre degradability decreased with the HCdiet, but increased with 2 MBsupplementation. Crude protein degradability increased with the HCdiet or 2 MBsupplementation. Abundance of Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenesand Bufyrivibrio fibrisolvensas well as activities of carboxymethyl cellulase, cellobiase, xylanase and pectinase decreased with the HCdiet, but increased with 2 MBsupplementation. However, abundance of Prevotella ruminicolaand Ruminobacter amylophilusas well as activities of α‐amylase and protease increased with the HCdiet or 2 MBsupplementation. Total PDexcretion also increased with the HCdiet or 2 MBsupplementation. The results suggested that growth performance, ruminal fermentation, CPdegradability and total PDexcretion increased with increasing dietary concentrate level from 40% to 60% or 2 MBsupplementation. The observed diet × 2 MBinteraction indicated that supplementation of 2 MBwas more efficacious for improving growth performance, ruminal fermentation and total PDexcretion with promoted ruminal bacteria abundance and enzyme activity in the MCdiet than in the HCdiet.