The Pearl River Source Region (PRSR), a fragile karst ecosystem in southwestern China, faces severe biodiversity threats from habitat fragmentation and rocky desertification driven by extensive economic development. This study constructs and evaluates the Ecological Security Pattern (ESP) for the PRSR to identify critical conservation elements and strategies. By integrating ecosystem service and the Minimum Cumulative Resistance (MCR) model, 133 ecological corridors were mapped among 57 ecological sources. Key network elements (patches, corridors, pinch points) were further evaluated using Circuit Theory (cumulative current centrality) and Graph Theory indices (dIICconnector, dPCconnector): Five patches (P14, P22, P24, P43, P49) were identified as extremely important sources, with the Pearl River Source Nature Reserve (P43, dIIC = 1.40, dPC = 4.82; P49, dIIC = 1.15, dPC = 3.49) emerging as the network core, Zhanyi Tiankeng Group (P42, dIIC = 0.30, dPC = 0.37) as a secondary hub. Crucially, corridor 42-49 sustains landscape connectivity in human-impacted areas, acting as a conduit for species migration and genetic exchange between tiankeng, the Pearl River Source, and distant regions, effectively reducing extinction risks. Besides, connectivity challenges were pronounced: western corridors exhibited high resistance due to long distances and intensive human pressure (agriculture, industry), while eastern corridors, though shorter, contained dense ecological pinch points (bottlenecks) exacerbated by transportation infrastructure and farmland encroachment. To mitigate fragmentation and enhance biodiversity conservation, this study proposes: (1) Strict protection of key sources via ecological redlines and reserve boundary optimization; (2) Restoration of priority corridors through vegetation rehabilitation and stepping-stone habitats; (3) Pinch points mitigation via wildlife-plant crossings and farmland-to-wetland conversion; (4) Economic-ecological coordination through eco-compensation, native broadleaved reforestation, and eco-industry development. This integrated ESP framework provides a replicable model for conserving biodiversity in globally vulnerable karst regions.