Inertial microfluidics uses the intrinsic fluid inertia in confined channels to manipulate the particles and cells in a simple, high-throughput, and precise manner. Inertial focusing in a straight channel results in several equilibrium positions within the cross sections. Introducing channel curvature and adjusting the cross-sectional aspect ratio and shape can modify inertial focusing positions and can reduce the number of equilibrium positions. In this work, we introduce an innovative way to adjust the inertial focusing and reduce equilibrium positions by embedding asymmetrical obstacle microstructures. We demonstrated that asymmetrical concave obstacles could break the symmetry of original inertial focusing positions, resulting in unilateral focusing. In addition, we characterized the influence of obstacle size and 3 asymmetrical obstacle patterns on unilateral inertial focusing. Finally, we applied differential unilateral focusing on the separation of 10- and 15-μm particles and isolation of brain cancer cells (U87MG) from white blood cells (WBCs), respectively. The results indicated an excellent cancer cell recovery of 96.4% and WBC rejection ratio of 98.81%. After single processing, the purity of the cancer cells was dramatically enhanced from 1.01% to 90.13%, with an 89.24-fold enrichment. We believe that embedding asymmetric concave micro-obstacles is a new strategy to achieve unilateral inertial focusing and separation in curved channels.