Human studies have established a positive association between the intake of industrial trans fatty acids and the development of cardiovascular diseases, leading several countries to enact laws that restrict the presence of industrial trans fatty acids in food products. However, trans fatty acids cannot be completely eliminated from the human diet since they are also naturally present in meat and dairy products of ruminant animals. Moreover, bans on industrial trans fatty acids have not yet been instituted in all countries. The epidemiological evidence against trans fatty acids by far overshadows mechanistic insights that may explain how trans fatty acids achieve their damaging effects. This review focuses on the mechanisms that underlie the deleterious effects of trans fatty acids by juxtaposing effects of trans fatty acids against those of cis-unsaturated fatty acids and saturated fatty acids (SFAs). This review also carefully explores the argument that ruminant trans fatty acids have differential effects from industrial trans fatty acids. Overall, in vivo and in vitro studies demonstrate that industrial trans fatty acids promote inflammation and endoplasmic reticulum (ER) stress, although to a lesser degree than SFAs, whereas cis-unsaturated fatty acids are protective against ER stress and inflammation. Additionally, industrial trans fatty acids promote fat storage in the liver at the expense of adipose tissue compared with cis-unsaturated fatty acids and SFAs. In cultured hepatocytes and adipocytes, industrial trans fatty acids, but not cis-unsaturated fatty acids or SFAs, stimulate the cholesterol synthesis pathway by activating sterol regulatory element binding protein (SREBP) 2-mediated gene regulation. Interestingly, although industrial and ruminant trans fatty acids show similar effects on human plasma lipoproteins, in preclinical models, only industrial trans fatty acids promote inflammation, ER stress, and cholesterol synthesis. Overall, clearer insight into the molecular mechanisms of action of trans fatty acids may create new therapeutic windows for the treatment of diseases characterized by disrupted lipid metabolism.