The osteogenic effect of culturing adipose-derived stem cells (ADSCs) on alendronate (Aln)-loaded polycarprolactone (PCL) nanofibrous scaffolds was evaluated by examining alkaline phosphatase (ALP) activity, calcium content, and expression of osteogenic differentiation genes in vitro. The 10% Aln/PCL nanofibrous scaffolds showed more ALP activity, mineralization, and osteocalcin and osteopontin mRNA than the 1% or 5% Aln/PCL nanofibrous scaffolds. The capacity of Aln/PCL nanofibrous scaffolds to regenerate new bone was studied in a rat calvarial defect model. New bone formation in vivo was evaluated by radiography, micro-computed tomography, and histological analysis. At 8 weeks after implantation, Aln/PCL scaffolds had a positive effect on bone regeneration and matrix formation. These results suggested that Aln/PCL nanofibrous scaffolds enhanced the osteogenic differentiation of ADSCs in vitro and bone formation in vivo.