Aim: To investigate negative voltage electrospinning of fibrous nanocomposite scaffolds bearing negative electric charges (N-poled), and determine whether and how retained negative charges could influence the biological performance of scaffolds for bone tissue engineering. Materials & methods: Poly(D,L-lactic acid) was used as the polymer matrix and carbonated hydroxyapatite nanospheres were the osteoconductive phase in the electrospun nanocomposite scaffolds. N-poled nanocomposite scaffolds were formed using negative voltage electrospinning, while conventional positive voltage electrospinning produced fibrous nanocomposite scaffolds bearing positive electric charges (P-poled). N-poled and P-poled scaffolds were systematically characterized and their biological performance was investigated through in vitro cell culture experiments. Results & conclusion: N-poled and P-poled scaffolds retained charges for different periods of time after electrospinning. Both types of scaffolds supported cell spreading and promoted filopodia formation. Compared with P-poled scaffolds, N-poled scaffolds enhanced cell proliferation, alkaline phosphatase activity and mineralization. N-poled scaffolds offer distinct advantages for bone tissue engineering.