The onset of insulin resistance (IR) in fresh dairy cows, will promote sparing of glucose, but the ability of insulin to inhibit HSL and suppress NEFA release from adipose may be impaired. The aim of this study was to evaluate the effects of feeding different FA in dairy cows on IR parameters. Twenty-four multiparous Iranian Holstein cows were used −30 d to 50 DIM. Dietary treatments consisted of (1) Prilled Palm (PO) [Energizer RP10, 2 and 2.25% DM in pre- and postpartum]; (2) Ca-salts of sunflower oil [Persia Fat-SO]; (3) Ca-salts of fish oil [Persia Fat- FO] and (4) equal amounts of Persia Fat-FO and Persia Fat-SO (2.2 and 2.5% of dietary DM in pre- and postpartum). Cows were weighed on 2 consecutive days to determine the doses of glucose for IVGTT(20 DIM, by administering 0.25 g/kg BW glucose i.v.) and insulin for insulin challenge (by 0.1 IU/kg BW insulin i.v. followed by saline). Blood samples were collected at −30 to180 min relative to administration of glucose and 120 min to insulin. The areas under the curve (AUC) of glucose and insulin during IVGTT and IC were calculated using the trapezoidal method. The parameters obtained from IVGTT and IC, were analyzed using PROC MIXED of SAS. The model included the fixed effects of treatment and sequence, and the random effects of period and cow within sequence. A covariate was used to adjust for differences in glucose concentration before the challenges. Statistically significant lower glucose and higher Insulin concentration in PO fed cows, in line with higher TNF-α and glycerol levels can be a hint to lower insulin sensitivity compared with Persia Fat fed animals. Accordingly, PO impaired glucose and NEFA CR (%/min) during IVGTT and glucose, insulin and NEFA during IC, reflecting lower responsiveness to insulin. The highest and lowest AUC (mg/dL) for glucose, NEFA and insulin during IVGTT, determined for PO and Persia Fat- Fish fed animals, respectively. After IC data support lower response to insulin in the case of glucose, NEFA and Insulin according to lower clearance rate for PO fed animals than Persia Fat. NEFA AUC30 (mEq/L) values were −13817 for PO vs. −16338, −18275 and −19462 for SO, Mix and FO. Overall, data support the idea that feeding rumen protected PUFA can modulate insulin resistance in fresh dairy cows.