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• Unsaturated fatty acids (UFA) supplementation with or without chitosan decreased methane. • Chitosan (CHI) associated with UFA did not influence the ruminal digestibility of nutrients. • Ruminal fermentation was altered with UFA supplementation associated with CHI. • UFA sources associated with CHI alter reticular flows of nutrients. • Chitosan associated UFA change the ruminal kinetics of beef heifers. This study evaluated the effects of supplementation of chitosan and unsaturated fatty acid (UFAs) sources on intake, digestibility, ruminal and nitrogen metabolism, blood metabolites, and feeding behavior of beef heifers. Five crossbred Simental x Zebu heifers (335 ± 53.2 kg of initial BW), ruminally cannulated, were randomly assigned according to a 5 × 5 Latin square design containing the following treatments: 1) CON, the control diet without supplementation; 2) SO, control diet supplemented with 25.3 g/kg of soybean oil; 3) WRS, control diet supplemented with 160 g/kg of whole raw soybean (WRS); 4) SOCHI, SO diet plus 14 g/kg of CHI; 5) WRSCHI, WRS diet plus 14 g/kg of CHI. No interaction effects between CHI and WRS were observed on nutrient intakes and digestibilities. Heifers fed SO, or WRS diets with or without CHI had lower enteric methane (CH 4) emissions (P < 0.01) than those fed CON. Moreover, animals fed SO and WRS had higher CH 4 emissions than those fed SOCHI and WRSCHI. No interaction between CHI and WRS was observed on nitrogen balance. However, heifers fed SO or WRS diets with or without CHI had lower nitrogen (N) intake (P = 0.001) and fecal N (P = 0.015) than those fed CON. Besides, animals fed SO and WRS diets showed lower retained N (P = 0.010) and efficiency (P = 0.005) than those fed SOCHI and WRSCHI. Heifers fed SO, or WRS diets with or without CHI showed lower rumination efficiencies of dry matter (DM; P < 0.01) and neutral detergent fiber (NDF; P = 0.013) than those fed CON. Heifers fed SO or WRS with or without CHI, showed lower reticular flows of DM (P = 0.049), NDF (P = 0.002), and potentially digestible NDF (P = 0.002) than those fed CON. In addition, animals fed SO or WRS diets with or without CHI showed a lower rumen digestion rate of DM, turnover rates of DM and NDF, and passage rates of DM and pdNDF than those fed CON. The supply of CHI to beef heifers fed diets with SO and WRS alters ruminal fermentation, methane production, nitrogen balance, feeding behavior, and ruminal dynamics. Nevertheless, the results strengthen the evidence that the provision of CHI in beef cattle diets can positively influence the effect of fatty acid sources. Besides, fatty acids sources provided alone have a negative influence on most metabolism parameters in beef heifers. [ABSTRACT FROM AUTHOR]