A backside illuminated image sensor includes pixel regions disposed in a substrate, an insulating layer disposed on a frontside surface of the substrate, a bonding pad disposed on a frontside surface of the insulating layer, and an anti-reflective layer disposed on a backside surface of the substrate. The substrate has a first opening for partially exposing a backside surface of the insulating layer, and the insulating layer has a third opening for partially exposing a backside surface of the bonding pad. The anti-reflective layer comprises a first portion disposed on an inner side surface of the first opening and a second portion disposed on a portion of the backside surface of the insulating layer exposed by the first opening and having a second opening connecting the first opening with the third opening, and a first spacer is disposed on inner side surfaces of the second opening and the third opening.