The sphingolipid ceramide (Cer) mediates the development of insulin resistance. Lipidomics has revealed that lignoceroyl sphingosine (C24:0-Cer) is a circulating biomarker for insulin resistance in dairy cattle. Environmental heat stress conditions compromise milk production, a response that may involve enhanced insulin action. Our objective was to investigate the effects of heat stress on circulating ceramide concentrations. Twelve multiparous, lactating Holstein dairy cows were assigned to 2 environmental conditions [thermoneutral (TN) or heat stress (HS)] for 7 d in a crossover design. Temperature-humidity index was maintained below 66 for TN treatment, and above 68 (peaking at 76) for HS treatment. Blood was collected at 0800 (AM) and 1900 h (PM) on d 6 and 7 of conditioning, and plasma samples pooled to reflect AM and PM metabolic status. Plasma concentrations of Cer, monohexosylceramide (GlcCer), and lactosylceramide were determined using mass spectrometry. Data were analyzed using a mixed model with fixed effects of treatment (HS and TN) and time (AM and PM). As previously established, heat stress increased rectal temperature and respiration rate, and reduced DM intake and milk production (P < 0.05). Circulating free fatty acids were elevated during AM, relative to PM (P < 0.05). Circulating β-hydroxybutyrate was increased by HS, relative to TN (P < 0.05). Relative to TN, HS did not increase C24:0-Cer or C24:0-dihydroceramide. Mild reductions in GlcCer levels were observed in response to HS treatment (e.g., 20% C20:0-GlcCer, P < 0.05), while lactosylceramide levels were unchanged. In contrast, C16:0-Cer and C16:0-dihydroceramide levels increased 14 and 19%, respectively. Plasma fatty acid levels were moderately associated with the majority of Cer quantified (r = 0.3 - 0.4; P < 0.05). For instance, C24:0-Cer was positively associated with circulating fatty acids (r = 0.38; P < 0.05). We conclude that short-term heat stress conditioning did not increase the insulin resistance biomarker C24:0-Cer. Our results suggest insulin resistance likely did not develop in heat-stressed cows.