The present study evaluated the interaction effects of n-3 fatty acids (from linseed oil (LSO), 0 v. 2·5, % DM basis) with rumen undegradable:degradable protein (RUP:RDP) ratios (low ratio (LR) 27:73; high ratio (HR) 38:62 based on crude protein %) in dairy calves' starter diet. Forty-eight 3-d-old female Holstein dairy calves (41·5 kg of body weight (BW)) were allocated in a 2 × 2 factorial arrangements in the following treatments (n 12 calves/each): (1) no supplementation of LSO with LR (NLSO-LR); (2) no supplementation of LSO with HR (NLSO-HR); (3) supplementation of LSO with LR (LSO-LR) and (4) supplementation of LSO with HR (LSO-HR). The calves were weaned on day 53 of the experiment and remained in the study until day 73. Intake was not affected by LSO and RUP:RDP ratio. However, average daily gain (ADG) was improved with LSO supplementation. Feeding the HR diet increased ADG compared with the LR diet during the entire period. Final BW was greater in calves fed on the LSO than those fed the NLSO diet. Microbial protein production did not differ among treatments. Calves fed on LSO diets had greater feed efficiency than those which were not fed on LSO diets. The calves supplemented with LSO had greater wither and hip heights compared with the unsupplemented calves. The glucose, cholesterol, HDL and insulin concentrations increased in calves supplemented with LSO. In conclusion, the HR diet improved calves' performance post-weaning; however, LSO could enhance growth performance of dairy calves during the pre-weaning period.