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To develop and validate a three-dimensional (3D) brain phantom that can be incorporated into existing stereotactic headframes to simulate stereotactic brain biopsy (SBB) and train veterinary surgeons.Experimental study.Canine brain phantoms were fabricated from osteological skull specimens, agarose brain parenchyma, and cheddar and mozzarella cheese molds (simulating meningiomas and gliomas).The neuroradiologic and viscoelastic properties of phantoms were quantified with computed tomography (CT) and oscillatory compression tests, respectively. Phantoms were validated by experienced and novice operators performing SBB on phantoms containing randomly placed, focal targets. Target yield and needle placement error (NPE) were compared between operators.Phantoms were produced in <4 hours, at an average cost of $92. The CT appearances of the phantom skull, agarose, and cheese components approximated the in vivo features of skull, brain parenchyma, and contrast-enhancing tumors of meningeal and glial origin, respectively. The complex moduli of the agarose and cheeses were comparable to the viscoelastic properties of in vivo brain tissues and brain tumors. The overall diagnostic yield of SBB was 88%. Although NPE did not differ between novice (median 3.68 mm; range, 1.46-14.54 mm) and experienced surgeons (median 1.17 mm, range, 0.78-1.58 mm), our results support the relevance of the learning curve associated with the SBB procedure.This 3D phantom replicates anatomical, CT, and tactile features of brain tissues and tumors and can be used to develop the technical skills required to perform SBB.