Strontium-containing hydroxyapatite (SrHA) is a promising material for bone repair and bone replacement due to the similar inorganic components with natural bone. In this study, the poly(epsilon-caprolactone) (PCL)/SrHA composite scaffold was fabricated by 3D printing method. Scanning electron microscopy (SEM) images of the fabricated scaffolds showed that SrHA was uniformly embedded in the interior of scaffold struts, and in vitro release profiles revealed that Sr and Ca ions released from the PCL/SrHA scaffold in a sustained manner. To confirm the performance of the fabricated composite scaffolds for bone regeneration, the cell proliferation and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs) grown on the scaffolds were evaluated. The experimental results indicated that incorporation of SrHA in the 3D printed PCL scaffold significantly facilitated the cell proliferation, and the PCL/SrHA scaffolds induced higher levels of BMSCs differentiation compared to the PCL and PCL/HA scaffolds, as demonstrated by ALP activity and osteo-related gene expression. Furthermore, in vivo cranial defect experiments further revealed that the incorporation of SrHA into 3D printed PCL scaffold was capable of promoting bone regeneration. Taken together, these results indicate that the PCL/SrHA composite scaffold can be readily fabricated by 3D printing technology and is highly promising as implantable material for bone tissue engineering application.