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The inhibition of resveratrol on the formation of thermally induced trans fatty acids in peanut oil and its structure-activity relationship were studied by gas chromatography (GC), liquid chromatography (LC), and theoretical calculation. The results showed that the content of resveratrol decreased with an increase in temperature, and too high temperatures caused a cut-off effect. Resveratrol significantly inhibited the formation of trans fatty acids in peanut oil, and the inhibition rate of total trans fatty acids decreased with increasing temperature. The highest inhibition rate of cis-trans isomerization of fatty acids was 30.30%. The inhibitory effect could be attributed to the energy barrier formed by reducing the rate of isomerization reaction and increasing the amount of trans fatty acids. Its quantitative parameters (intrinsic thermodynamic energy and total entropy) determined the anti-isomerization effect. Moreover, the structure-activity relationship for the anti-isomerization effect was established, which will lay a theoretical foundation for the precise regulation of trans fatty acids.