Excessive rates of lipolysis during periods of negative energy balance (NEB) are associated with increased susceptibility to disease. Lipolysis increases adipose tissue macrophage (ATM) populations. Depending on their phenotype, ATMs modify inflammatory processes and alter adipose metabolic functions. Classically activated ATMs (M1) are pro-inflammatory while alternatively activated ATMs (M2) promote inflammation resolution. The objective of this study was to evaluate changes in ATM trafficking and phenotype in healthy cows during feed restriction-induced NEB. Lactating multiparous dairy cows (DIM 119–210) were fed a common diet to meet nutrient requirements during a 14d preliminary period (d1 to 14) and then randomly assigned to one of 2 feeding protocols: ad libitum (AL; n = 6) or feed-restricted (FR; n = 7). Caloric intake was reduced in FR cows for 4 d (d15 to 18) to achieve a targeted NEB of −15 Mcal/d. Omental and subcutaneous adipose tissue samples were collected to harvest stromal vascular cells (SVC) on d11 and d18. Data were analyzed in a mixed model with treatment and day as fixed effects and cow as a random effect. FR cows reached a NEB of −13.5 ± 1.9 Mcal/d inducing a lipolytic response (NEFA on d18: FR = 0.52 mEq/L; AL = 0.17 mEq/L; P < 0.01), while AL animals remained in positive energy balance (3.2 ± 2.2 Mcal/d). Flow cytometry analysis revealed that at d18, FR increased the infiltration ratio of CD68⁺, a specific ATM surface marker, in omental SVC (FR = 2.85 ± 0.39; AL = 1.25 ± 0.35; P = 0.01), while the expression ratio of CD14, an M1 marker, remained unaltered (P = 0.86). Adipose tissue from FR cows exhibited an increased expression of the macrophage-related gene SIRPA (P = 0.01), but no change in M1 genes CCL2 and TNFα (both P > 0.49). Additionally, compared with AL, FR upregulated the expression of M2 specific genes IL10 and ARG1 (both P < 0.01). This finding contrasts with the predominately M1 phenotype observed previously in ATMs from clinically diseased cows. These results provide evidence for an active role of ATMs during NEB in ruminants and emphasize changes in their inflammatory phenotype during lipolytic periods.