Toward the goal of establishing physiologically relevant in vitro tumor models, we synthesized and characterized a biomimetic hydrogel using thiolated hyaluronic acid (HA-SH) and an acrylated copolymer carrying multiple copies of cell adhesive peptide (PolyRGD-AC). PolyRGD-AC was derived from a random copolymer of tert-butyl methacrylate (tBMA) and oligomeric (ethylene glycol) methacrylate (OEGMA), synthesized via atom transfer radical polymerization (ATRP). Acid hydrolysis of tert-butyl moieties revealed the carboxylates, through which acrylate groups were installed. Partial modification of the acrylate groups with a cysteine-containing RGD peptide generated PolyRGD-AC. When PolyRGD-AC was mixed with HA-SH under physiological conditions, a macroscopic hydrogel with an average elastic modulus of 630 Pa was produced. LNCaP prostate cancer cells encapsulated in HA-PolyRGD gels as dispersed single cells formed multicellular tumoroids by day 4 and reached an average diameter of ∼95 μm by day 28. Cells in these structures were viable, formed cell-cell contacts through E-cadherin (E-CAD), and displayed cortical organization of F-actin. Compared with the control gels prepared using PolyRDG, multivalent presentation of the RGD signal in the HA matrix increased cellular metabolism, promoted the development of larger tumoroids, and enhanced the expression of E-CAD and integrins. Overall, hydrogels with multivalently immobilized RGD are a promising 3D culture platform for dissecting principles of tumorigenesis and for screening anticancer drugs.