Osteochondral regeneration remains formidable challenges despite significant advances in microsurgery. Herein, an acellular trilayer cryogel (TC) with injectability, tunable pore sizes (80–200 µm), and appropriate compressive modulus (10.8 kPa) is manufactured from self‐healable hydrogel under different gelling times through Schiff reaction between chitosan and difunctionalized polyurethane (DFPU). Bioactive molecules (Y27632 and dexamethasone) are respectively loaded in the top and bottom layers to form the Y27632/dexamethasone‐loaded trilayer cryogel (Y/DEX‐TC). Mesenchymal stem cells (MSCs) seeded in Y/DEX‐TC proliferated ≈350% in vitro and underwent chondrogenesis or osteogenesis in response to the respective release of Y or DEX in 14 days. Acupuncture is administered to animals in an attempt to modulate the innate regulatory system and mobilize endogenous MSCs for osteochondral defect regeneration. In vivo rabbit experiments using Y/DEX‐TC combined with acupuncture successfully regulate SDF‐1 and TGF‐β1 levels, which possibly cause MSC migration toward Y/DEX‐TC. The synergistic effect of cryogel and acupuncture on immunomodulation is verified with a ≈7.3‐fold enhancement of the M2‐/M1‐macrophage population ratio by treatment of Y/DEX‐TC combining acupuncture, significantly greater than ≈1.5‐fold increase by acupuncture or ≈2.2‐fold increase by Y/DEX‐TC alone. This novel strategy using acellular drug‐loaded cryogel and accessible acupuncture shows promise in treating osteochondral defects of joint damage.