The objective was to investigate the relationship between elevated plasma nonesterified fatty acid (NEFA) concentration and insulin resistance in Holstein cows. Six nonlactating, nongestating, ruminally cannulated Holstein cows were blocked by body condition score and randomly assigned to a sequence of 2 treatments in a crossover design. Cows were offered legume and grass hay ad libitum supplemented with minerals and vitamins and were allowed free access to water and a trace mineralized salt block. Mobilization of body reserves was stimulated by withdrawing forage for 48 h before initiation of treatments. Treatments consisted of 11 hourly abomasal infusions of water (control) or nicotinic acid (NA; 6 mg/h per kg of body weight) as an antilipolytic agent. Infusions of NA decreased plasma NEFA concentration from 545 {micro}Eq/L to approximately 100 {micro}Eq/L within 2 h after initiation of treatments, and differences were maintained throughout infusions. Intravenous glucose tolerance test was performed 8 h after initiation of treatments and was followed by 3 h of blood sampling. The reduction of plasma NEFA concentration led to significantly greater glucose clearance rate (1.9 vs. 1.2%/min) and to decreased glucose half-life (37 vs. 58 min), time to reach basal concentration (81 vs. 114 min) and glucose response area under the curve during 180 min of sampling [6,942 vs. 10,085 ({micro}IU/mL) x 180 min]. Enhanced glucose clearance was achieved when plasma NEFA was reduced by NA, despite lower insulin concentration (70.0 vs. 97.9 {+/-} 13.4 {micro}IU/mL) and a tendency for smaller insulin response area under the curve during 180 min of sampling [7,646 vs. 12,104 {+/-} 2,587 ({micro}IU/mL) x 180 min], reflecting an increased response to endogenous insulin. Based on literature, we do not expect NA to have altered glucose metabolism directly; therefore, this experiment demonstrates a cause and effect relationship between elevated NEFA and insulin resistance in Holstein cows.