Compared with cemented implants in total knee arthroplasty (TKA), cementless prostheses can provide better biological fixation for young and active patients by their osseointegration with host bone. However, to date, early loosening occurs in most cementless knee prostheses due to extensive bone ingrowth deficiencies, especially at the tibial bone-prosthesis interfaces. This study aims to improve the osseointegration capability of 3D-printed cementless tibial prostheses by modifying metal surfaces with a three-partition trabecular-like porous structure. Ti6Al4V tibial prostheses were prepared with a novel three-partition trabecular surface design by electron beam melting (EBM) according to the regional structural and mechanical properties of human tibial plateaus. The trabecular prostheses were implanted in the knee joints of sheep in situ to evaluate the osseointegration and stability of the designed tibial implants. X-ray images and anatomical observation showed no detachment or displacement of the trabecular implants at 3 and 6 months postoperation. Micro-CT evaluation and histological analysis showed improved osseointegration and strengthened biological fixation of the trabecular implants. The cellular experiment results indicated that three topological types of porous scaffolds corresponding to the three-partition trabecular prosthesis design were favorable for the proliferation and differentiation of preosteoblasts. In conclusion, a novel three-partition trabecular surface design enhanced the osseointegration capacity of 3D-printed Ti6Al4V tibial implants, which will promote the application of cementless knee prostheses in clinics.