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Palmitic (C16:0), α-linolenic acid (C18:3n-3 <i>cis</i>), and propionate regulate bovine pyruvate carboxylase (<i>PC</i>) and phosphoenolpyruvate carboxykinase (<i>PCK1</i>) expression in vitro. The objective of this experiment was to determine the impact of C16:0, C18:3n-3 <i>cis</i>, propionate, and acetate postruminal infusions on hepatic <i>PC</i> and <i>PCK1</i> expression. We hypothesized that circulating fatty acids alter hepatic <i>PC</i> and <i>PCK1</i> in lactating dairy cows. Acetate, propionate, palm oil, and flaxseed oil were supplied postruminally to lactating cows (<i>n</i> = 4) using two 4 × 4 Latin square studies. For Experiment 1, cows were infused on an hourly basis with either a bolus of propionate, acetate, or the combination of propionate and palm oil, or acetate and palm oil, and Experiment 2 was similar, but flaxseed oil replaced palm oil. Flaxseed infusions increased plasma concentration and the molar percent of C18:3n-3 <i>cis</i> and decreased C16:0 but did not affect <i>PC</i> or <i>PCK1</i> expression. Palm infusions did not affect blood metabolites or the hepatic expression of <i>PC</i> or <i>PCK1</i>. The lack of responses to short-chain fatty acid infusions and changes in circulating long-chain fatty acids in mature cattle are not suitable models to study the effects of α-linolenic acid and propionate on bovine <i>PC</i> and <i>PCK1</i> expression previously observed in vitro.