With an increasing demand for adhesives, the durability of joints has become highly important. The fatigue resistance of adhesives has been investigated mainly for epoxies, but in recent years many other resins have been adopted for structural adhesives. Therefore, understanding the fatigue characteristics of these resins is also important. In this study, the cyclic fatigue behavior of a two-part acrylic-based adhesive used for structural bonding was investigated using a fracture-mechanics approach. Fatigue tests for mode I loading were conducted under displacement control using double cantilever beam specimens with varying bond-line thicknesses. When the fatigue crack growth rate per cycle, da/dN, reached 10−5 mm/cycle, the fatigue toughness reduced to 1/10 of the critical fracture energy. In addition, significant changes in the characteristics of fatigue crack growth were observed varying the bond-line thickness and loading conditions. However, the predominance of the adhesive thickness on the fatigue crack growth resistance was confirmed regardless of the initial loading conditions. The thicker the adhesive bond line, the greater the fatigue toughness.