Hydrogels can provide a controllable cell microenvironment for numerous applications in regenerative medicine, and delivery of gene therapy vectors can be employed to enhance their bioactivity. We investigated the delivery of lentiviral vectors from hydrogels, and employed the immobilization of lentivirus to hydroxylapatite (HA) nanoparticles as a means to retain and stabilize vectors within hydrogels, and thereby increase delivery efficiency. Entrapment of the vector alone within the hydrogel maintained the activity of the virus more effectively compared to the absence of a hydrogel, and release was slowed with an increasing solid content of the hydrogel. Association of the lentivirus with HA increased the activity of the complexes, with HA increasing the virus activity for 72 hours. Cells seeded onto lentivirus-HA-loaded hydrogels had a decreased number of infected cells outside of the hydrogel relative to the absence of HA. In vivo studies with collagen hydrogels loaded with lentivirus and HA-lentivirus demonstrated sustained and localized transgene expression for at least 4 weeks, with increased expression using the lentivirus-HA complex. This strategy of nanoparticle immobilization to stabilize and retain vectors is broadly applicable to hydrogels, and may provide a versatile tool to combine gene therapy and biomaterials for applications in regenerative medicine.