Due to tissue lineage variances and the anisotropic physiological characteristics, regenerating complex osteochondral tissues (cartilage and subchondral bone) remains a great challenge, which is primarily due to the distinct requirements for cartilage and subchondral bone regeneration. For cartilage regeneration, a significant amount of newly generated chondrocytes is required while maintaining their phenotype. Conversely, bone regeneration necessitates inducing stem cells to differentiate into osteoblasts. Additionally, the construction of the osteochondral interface is crucial. In this study, we fabricated a biphasic multicellular bioprinted scaffold mimicking natural osteochondral tissue employing three‐dimensional (3D) bioprinting technology. Briefly, gelatin‐methacryloyl (GelMA) loaded with articular chondrocytes and bone marrow mesenchymal stem cells (ACs/BMSCs), serving as the cartilage layer, preserved the phenotype of ACs and promoted the differentiation of BMSCs into chondrocytes through the interaction between ACs and BMSCs, thereby facilitating cartilage regeneration. GelMA/strontium‐substituted xonotlite (Sr‐CSH) loaded with BMSCs, serving as the subchondral bone layer, regulated the differentiation of BMSCs into osteoblasts and enhanced the secretion of cartilage matrix by ACs in the cartilage layer through the slow release of bioactive ions from Sr‐CSH. Additionally, GelMA, serving as the matrix material, contributed to the reconstruction of the osteochondral interface. Ultimately, this biphasic multicellular bioprinted scaffold demonstrated satisfactory simultaneous regeneration of osteochondral defects. In this study, a promising strategy for the application of 3D bioprinting technology in complex tissue regeneration was proposed.