Chemical and physical treatments of barley grain increase ruminally resistant starch and can improve the rumen fermentation pattern. The objective of the present study was to evaluate the effects of chemical (addition of citric acid, CA) and physical (grinding to two different particle sizes, PS) treatment of barley grain on performance, rumen fermentation, microbial protein yield in the rumen and selected blood metabolites in growing calves. In all, 28 male Holstein calves (172±5.1 kg initial BW) were used in a complete randomised design with a factorial arrangement of 2 barley grain particle sizes×2 levels of citric acid. The diets were as follows: (i) small PS (average 1200 µm) barley grain soaked in water (no CA addition); (ii) small PS barley grain soaked in a CA solution (adding 20 g CA/kg barley); (iii) large PS (average 2400 µm) barley grain soaked in water (no citric acid addition) and (iv) large PS barley grain soaked in a citric acid solution (adding 20 g CA/kg barley). Barley grain was then incorporated at 35% in a total mixed ration and fed to the calves for 11 weeks. Feeding small PS barley decreased feed intake (P=0.02) and average daily weight gain (P=0.01). The addition of CA to barley grain did not affect intake but increased weight gain (P<0.01) and improved feed to gain ratio (P=0.03). Digestibility of organic matter and NDF tended (P<0.10) to increase, whereas faecal scoring was improved (P=0.03) and the presence of undigested grain particles in faeces was reduced (P<0.01) with CA-treated barley grain. Glucose and urea concentrations were increased (P<0.01) in the blood of calves fed the CA-treated barley grain. Ruminal pH tended (P=0.08) to be decreased with more finely ground barley and was increased when barley grain was treated with CA. Total volatile fatty acid concentrations in the rumen did not differ among treatments (P>0.05). However, the molar proportion of propionate was increased (P=0.03) when barley was more finely ground, and that of acetate was increased (P=0.04) when CA was added to barley grain. The ruminal concentration of ammonia nitrogen was increased (P<0.01) and microbial nitrogen synthesis in the rumen tended to decrease by adding CA to barley. Treating barley grain with citric acid increased fibre digestibility of total mixed rations, attenuated the decrease in ruminal pH, and improved weight gain and feed efficiency in male Holstein growing calves fed a high-cereal diet (550 g cereal grain/kg diet).