trans-10,cis-12 conjugated linoleic acid (t10,c12-CLA) was linked to milk fat depression in dairy cows; transcription factor sterol response element binding protein-1 (SREBP1) regulates fatty acid synthesis. SREBP1 activation and migration to the nucleus requires the removal of Insig1, a protein that anchors SREBP1 in the endoplasmic reticulum membrane. The molecular basis orchestrating the effect of t10,c12-CLA on bovine SREBP1 activation has not yet been elucidated. We hypothesize that t10,c12-CLA reduces SREBP1 activation through delay of Insig1 degradation. In the present study, we employed a bovine mammary epithelial cell line (Mac-T) and found that, mRNA and protein levels of SREBP1 declined over 56% when cells were treated with 60 µM or greater t10,c12-CLA for 24 h (P < 0.05). Similar dose effects were observed in the mRNA expression of SREBP1-regulated genes including FAS, SCD1, and Insig1. Compared with 0 µM t10,c12-CLA, 60 µM or higher CLA increased Insig1 protein expression over 2-fold in cells transfected with FLAG-tagged Insig1 (P < 0.05). The effect was greater with t10, c12-CLA than other fatty acids including cis9, trans11-CLA, linoleic acid, or oleic acid when cells were treated with 75 µM for 6 h. Further investigation revealed that increased FLAG-Insig1 was due to the inhibitory effect of t10,c12-CLA on the proteasomal degradation of Insig1. Cells treated with 75 µM t10,c12-CLA or 10 µM MG132, a proteasome inhibitor, for 6 h had 2.5-fold greater accumulation of FLAG-Insig1 compared with 0 µM t10,c12-CLA (P < 0.05). The degradation of FLAG-Insig1 was delayed when cells were treated with 75 µM t10,c12-CLA for 6 h. These findings suggest that t10,c12-CLA plays a role in regulating SREBP1 activation by reducing proteasomal degradation of Insig1. We conclude that stabilized Insig1 retains SREBP1 in the ER preventing activation and migration to nucleus, thus reducing lipogenic gene transcription.