The present study evaluated the effects of linseed oil (LO) and two grain sources on growth performance, microbial protein yield (MPY), blood metabolites, and inflammatory markers in Holstein dairy calves. Forty-eight 3-day-old dairy calves (24 males and 24 females) with starting BW of 40.3 ± 1.6 kg were allocated in a completely randomized block design with a 2 × 2 factorial arrangement as follow; 1) Corn grain (CG) with no LO supplementation (CG-NLO), 2) CG with 2.5% LO supplementation (CG-LO), 3) Barley grain (BG) with no LO supplementation (BG-NLO), and 4) BG with 2.5% LO supplementation (BG-LO). The calves were weaned on d 59 but the study lasted for 14 d after weaning (d 73 of experiment). The results showed that starter feed intake was influenced neither by grain source nor linseed oil. However, average daily gain, BW, hip height, and MPY were improved in calves received BG compared to CG diets. Linseed oil supplementation had no significant effects on growth performance and MPY. During pre-weaning period, calves fed BG-LO had the greatest feed efficiency and the highest wither height. However, the greatest tumor necrosis factor and serum amyloid A were observed in BG-NLO. Despite, LO supplementation did not influence growth performance of animals per se; however, it reduced circulating inflammatory markers in calves during pre-weaning period. Based on the current study condition, BG is more favourable than CG in dairy calves from the daily gain and microbial protein synthesis perspectives, and supplementing the starters with n-3 FA can be strategy to improve immune performance of calves fed barley-based starter diets.