OBJECTIVE: The aim of this study was to determine the effect of rumen-protected rubber seed oil (RPRSO) supplementation on feed digestibility, milk yield, and milk composition in tropical dairy cows. METHODS: Twelve crossbred Holstien-Friesian dairy cows (75% Holstein-Friesian, 25% Thai native breed) with a mean body weight of 460±30 kg and 20±5 days in milk were randomly assigned to 1 of 3 treatments according to a completely randomized design. The treatments were as follows: a basal diet without rumen-protected fat (RPF) (control) or supplementation of rumen-protected palm oil (RPPO) at 300 g/h/d and RPRSO at 300 g/h/d. Each cow was fed a total mixed ration ad libitum. RESULTS: The nutrient intake was similar among treatments (p>
0.05). Adding RPF did not affect nutrient digestibility, while organic matter (OM) digestibility increased in dairy cows receiving RPRSO (p<
0.01). Blood urea nitrogen (BUN), total protein, or glucose did not alter among treatments (p>
0.05), while triglycerides and cholesterol were increased when cows were fed RPPO (p<
0.01). Adding RPF increases milk yield in cows (p<
0.01). The supplementation of RPRSO increased milk fat (p = 0.04). Milk fat yield was higher in RPPO and highest in RPRSO (p<
0.01). The addition of RPF increased the oleic acid (OA, C18:1 cis-9) in milk (p = 0.01). In addition, cows fed RPRSO increased linoleic acid (LA
C18:2ω6 cis-9,12+trans-9,12) and α-linolenic acid (ALA
C18-3ω3 cis-9,12,15) in milk (p<
0.01). The addition of RPF increased milk unsaturated fatty acids and monounsaturated fatty acids (p≤0.04). The polyunsaturated fatty acid (PUFA) in milk increased with RPRSO supplementation (p<
0.01). CONCLUSION: Supplementation of RPRSO during early lactation can increase feed digestibility and the concentration of milk fat with PUFA (LA and ALA) in tropical dairy cows.