The objective of this study was to evaluate the microbial protein synthesis of Jersey heifers supplemented with chitosan or omega-6 fatty acids (FA) source. Eight animals (average body weight of 158.62 ± 1.75 kg, mean ± SD) were used in replicated 4 × 4 Latin square experimental design, balanced and contemporary, in 2 × 2 factorial arrangements. The experimental period consisted of 18 d (12 d for adaptation and 6 d for data collection) and 5 d were used for wash out. The experimental diets were (1) control (CO), without omega-6 FA and chitosan supplementation; (2) whole raw soybean (WRS, source of omega-6 FA) with 200 g/kg of DM of WRS); (3) chitosan (CHI), with 2 g/kg of DM of chitosan; (4) chitosan and WRS (CHWS), with chitosan and WRS in the same level used in previously cited treatments. The diets were formulated according to the NRC (2001) to achieve weight gain of 700 g/day. Spot urine samples were collected on d 17 of each period 4 h after feeding. The analyses were performed according to Chen and Gomes (1992). Data were analyzed using PROC MIXED of SAS 9.3.Interaction effect (P < 0.05) was observed between CHI and WRS for uric acid (mmol/L), which CHI reduced (1.74 mmol/L) and CHIWS increased uric acid (3.34 mmol/L). Animals fed CHI presented decrease (P < 0.05) in total purine and absorbable purine derivatives. The output of microbial nitrogen and protein (g/d) was reduced (P < 0.05) when heifers were supplemented only with CHI. Animals fed CHI presented decrease in the output of microbial nitrogen and protein in 31.55, 35.53 and 15.07% when compared with CO, WRS and CHWS, respectively. The chitosan supplementation reduced the microbial protein synthesis of dairy Jersey heifers.