The ratio between sphingolipids is a means to understand sphingolipid biology, an analysis utilized in biomedicine to study ceramide-mediated insulin resistance. Our objective was to evaluate whether the ratio between plasma sphingolipids in dairy cows is modified during the peripartum. Multiparous Holstein cows were grouped by BCS at d −28 prepartum: lean (BCS 2.91 ± 0.13; n = 7) or overweight (OVER; BCS 4.03 ± 0.21; n = 7). Diets were formulated to meet nutrient requirements. Blood was collected routinely from d −21 to 21. LC/MS was used to profile 37 Cer, monohexosylceramides (GlcCer), lactosylceramides (LacCer), and SM in plasma. Log-transformed data were analyzed using a mixed model with repeated measures (fixed effects of BCS and day). Nonparametric correlations were analyzed. NEFA mobilization increased during transition, more so in OVER (P < 0.01). Ratio of C16:0-SM to C16:0-Cer (C16:0 SM:Cer) increased during transition (P < 0.01) and tended to be lower in OVER (P < 0.1). C18:0 SM:Cer reached a nadir at calving (P < 0.01). In contrast, C24:0 and C22:1 SM:Cer progressively decreased with time (P < 0.01). C16:0, C22:0, and C26:0 Cer:GlcCer decreased during transition (P < 0.01). C18:0 Cer:GlcCer tended to display a biphasic response (increased then declined; P = 0.08). Neither BCS nor day modified C24:0 Cer:GlcCer. C16:0 and C18:0 GlcCer:LacCer increased and decreased, respectively, as calving approached (P < 0.01). After calving, C24:0 GlcCer:LacCer declined until d 21 (P < 0.05). C18:0 and C24:0 SM:Cer, C16:0 and C20:0 Cer:GlcCer, and C18:0 and C24:1 GlcCer:LacCer were negatively correlated with NEFA (r = −48 to −0.22; P < 0.01). In contrast, C16:0 GlcCer:LacCer was positively correlated with NEFA (r = 0.57; P < 0.01). C18:0 and C26:0 Cer:GlcCer were negatively correlated with estimated insulin sensitivity (RQUICKI; r = −0.35 to −0.29; P < 0.01), whereas, C20:0 Cer:GlcCer and C18:0 GlcCer:LacCer were positively correlated with RQUICKI (r = 0.19 to 0.29; P < 0.05). Research will need to determine whether changes in plasma fatty acids are related to the ratio between acyl-chain specific sphingolipids.