Excess saturated fatty acids can impair mitochondrial β-oxidation in overweight monogastrics. In turn, long-chain acylcarnitines (LCAC) can accumulate in plasma, biomarkers for insulin resistance in humans. Our objective was to determine whether plasma LCAC are associated with changes in estimated insulin sensitivity in dairy cows. Our data included multiparous Holstein cows grouped according to BCS at d −30 prepartum: lean (BCS < 3.0; n = 10) or overweight (BCS > 4.0, n = 11; OVER), with blood collected at d −45, −30, −15, −7, and 4, relative to calving. Profiling of LCAC was achieved using LC/MS. Data were analyzed using a mixed model with repeated measures including the random effect of cow and the fixed effects of BCS and time. Nonparametric correlations were analyzed. We previously demonstrated that OVER had greater NEFA mobilization and lower insulin sensitivity at d −30, −15 and −7 prepartum (P < 0.05), relative to lean. LC/MS detected C14:0-, C16:0-, C18:0-, and C20:0-LCAC, representing 12, 31, 48, and 9% of total plasma LCAC, respectively. Concomitant with lipolysis, plasma C14:0-, C16:0-, C18:0, and C20:0-LCAC, and total LCAC increased (P < 0.01). Plasma C16:0- and C20:0-LCAC, and total LCAC levels were greater in OVER at d −7 (P < 0.05). Plasma C14:0- and C18:0-LCAC levels were higher in OVER (P = 0.10). A BCS × time interaction was observed, where OVER had greater plasma C18:0- and C20:0-LCAC levels at d −7 (P = 0.06 and P < 0.05, respectively). Plasma C14:0-, C16:0-, C18:0-, and C20:0-LCAC, and total LCAC were positively correlated with NEFA (P < 0.01). Utilizing previously acquired GC/MS data, circulating C16:0- and C18:0-LCAC were positively correlated with mobilized palmitic acid, stearic acid, and lipolysis marker glycerol (P < 0.01). Plasma C14:0-, C16:0-, C18:0, and C20:0-LCAC, and total LCAC were negatively correlated with insulin sensitivity (P < 0.01). Our data demonstrate greater plasma LCAC in insulin resistant, overweight periparturient dairy cows; however, the origin and involvement of LCAC in the progression of insulin resistance is unclear.