Ru–MgAl catalysts were prepared by co-precipitation at different pH values (x = 7–12), with the aims of investigating the effect of pH on their catalytic performance for isomerization of linoleic acid to conjugated linoleic acid and of understanding the relationship of basicity site and ruthenium activity site. The results showed that strong basicity and highly dispersed ruthenium may be the active sites for linoleic acid isomerization. Only if all three conditions, i.e., high Ru dispersion, appropriate SMSI effect and high number of strong basicity sites, are fulfilled can highly effective active sites be formed for the isomerization of linoleic acid to conjugated linoleic acid. A possible reaction mechanism for the isomerization was also proposed.