Different monounsaturated fatty acid (MUFA) species have distinct pathophysiological activities. cis-Palmitoleic acid (16:1n-7) was previously reported to improve insulin sensitivity in animal studies. The proportions of hepatic MUFAs are generally considered to reflect changes in the activities of fatty acid modifications (∆9 desaturation and fatty acid elongation). However, hepatic levels of 16:1n-7 are markedly lower than those of oleic acid (18:1n-9). Nevertheless, no convincing explanation has yet been provided for the low level of 16:1n-7. We hypothesized that fatty acid degradation plays a key role in maintaining a low 16:1n-7 proportion in the liver. In order to corroborate the link between β-oxidation and the proportion of 16:1n-7, rats were fed a control diet, fed a fat-free diet to up-regulate fatty acid modifications, but not β-oxidation, or treated with clofibric acid to up-regulate fatty acid modifications and β-oxidation. The nutritional manipulation markedly increased the proportions of 16:1n-7, 18:1n-9, and cis-vaccenic acid (18:1n-7). Although the pharmacological manipulation enhanced fatty acid modifications to largely the same extent as the nutritional manipulation and markedly elevated the proportion of 18:1n-9, those of 16:1n-7 and 18:1n-7 remained largely unchanged. The oxidation rates of 16:1n-7, 18:1n-9, and 18:1n-7 in liver slices were in the following order: 16:1n-7>18:1n-7≑18:1n-9 in control livers, and were increased by the pharmacological manipulation and decreased by the nutritional manipulation. These results strongly suggest that β-oxidation, in concert with fatty acid modifications, plays a key role in regulating the MUFA profile and is crucially involved in maintaining low 16:1n-7 levels in the liver.