The objective of this study was to evaluate the nitrogen balance 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 designs, 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 2g/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. Blood samples were collected on d 15 of each period before feeding, by puncture of coccygeal vein and immediately centrifuged at 2000 g × 15 min, supernatant serum was transferred do tubes and submitted to analyses. Spot urine samples were collected on d 17 of each period 4 h after feeding. For the evaluation of nitrogen compounds were dosed concentrations of urea and creatinine (mg/dL) in blood and urine by enzymatic colorimetric method. Data were analyzed using PROC MIXED of SAS 9.3.Animals fed CHI presented lower (P < 0.05) concentration of urea and higher (P < 0.05) concentrations of creatinine (0.80 mg/dL) in blood than other diets. Higher concentration of urea (43.12 mg/dL) was observed for heifers fed WRS and lower concentration of creatinine than others experimental diets. The renal clearance of urea (24 h period) presented highest value (P < 0.05) for animals fed WRS and lowest (P < 0.05) for animals fed CHI. Fractional urea excretion (%) was highest for animals fed CHI and lowest for animals fed WRS. Chitosan and omega 6 FA source supplementation altered the nitrogen balance of dairy Jersey heifers.