The combination of a sudden increase in metabolic demands and the reduced dry matter intake in early postpartum cows is regarded as the culprit of a sudden increase in nonesterified fatty acids (NEFA), indicative of the rapid mobilization of lipid reserves. We hypothesized that the increase in concentration of circulating NEFA would alter gene expression profiles via modulation of peroxisome proliferator-activated receptors (PPAR), transcription factors known to respond to fatty acids and with a well-defined role in metabolism and inflammation-associated gene regulation. To mimic the natural cellular environment, we ran a series of experiments using whole cow serum with different NEFA concentrations (0.41 mEq/L, SLN; 0.71 mEq/L, SHN) to treat a bovine mammary epithelial cell line (MAC-T) and human liver hepatocellular carcinoma cells (HepG2) using either a bioluminescent or a fluorescent reporter assay to assess PPAR activation and assess cell viability. Data were analyzed using general linear model procedure of SAS. Both SLN and SHN activated PPAR in MAC-T cells when compared with the control (P < 0.001) but these results were not recapitulated in HepG2. To assess directly the effect of NEFA, cells were treated with NEFA isolated from each serum and resuspended at equimolar concentration in BSA-free medium. NEFA in medium had high (P < 0.0001) cytotoxicity in both cell types. The cytotoxicity decreased as NEFA was spiked into the serum at decreasing concentration. Despite their clear detrimental effect on cellular viability, extracted NEFA had a dramatic effect on PPAR activation, especially in MAC-T (>3.5-fold; P < 0.001). To assess if NEFA released from VLDL activate PPAR, 2 ng/µL of bovine lipoprotein lipase (LPL) was added to the serum. The addition of LPL resulted in significant activation of PPAR in HepG2 cells (>2-fold; P < 0.001), although viability was negatively affected (P < 0.001), but not MAC-T cells. Our preliminary results clearly indicated that extracted NEFA, but also NEFA released by LPL, especially for HepG2, are cytotoxic. Despite this, our data indicated that NEFA stimulate PPAR activity, suggesting the possible involvement of the PPAR pathway in the metabolic response to the increasing circulating NEFA during the transition period.