Osteoporosis is a metabolic multifaceted disorder, characterized by insufficient bone strength. It has been recently shown that advanced glycation end products (AGEs) play a role in senile osteoporosis, through bone cell impairment and altered biomechanical properties. Pentosidine (PENT), a wellcharacterized AGE, is also considered a biomarker of bone fracture. Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D3, are prerequisites for optimal osteoblasts functioning. Vitamin K2 is known to enhance in vitro and in vitro vitamin D-induced bone formation. The aim of the study was to assess the effects of Vitamins D3 and K2 and PENT on in vitro osteoblast activity, to convey a possible translational clinical message. Ex vivo human osteoblasts cultured, for 3 weeks, with vitamin D3 and vitamin K2 were exposed to PENT, a well-known advanced glycoxidation end product for the last 72 hours. Experiments with PENT alone were also carried out. Gene expression of specific markers of bone osteoblast maturation [alkaline phosphatase, ALP; collagen I, COL Iα1; and osteocalcin (bone-Gla-protein) BGP] was measured, together with the receptor activator of nuclear factor kappa-B ligand/osteoproteregin (RANKL/OPG) ratio to assess bone remodeling. Expression of RAGE, a well-characterized receptor of AGEs, was also assessed. PENT+vitamins slightly inhibited ALP secretion while not affecting gene expression, indicating hampered osteoblast functional activity. PENT+vitamins up-regulated collagen gene expression, while protein secretion was unchanged. Intracellular collagen levels were partially decreased, and a significant reduction in BGP gene expression and intracellular protein concentration were both reported after PENT exposure. The RANKL/OPG ratio was increased, favouring bone reabsorption. RAGE gene expression significantly decreased. These results were confirmed by a lower mineralization rate. We provided in vitro evidence that glycoxidation might interfere with the maturation of osteoblasts, leading to morphological modifications, cellular malfunctioning, and inhibition of the calcification process. However, these processes may be all partially counterbalanced by vitamins D3 and K2. Therefore, detrimental AGE accumulation in bone might be attenuated and/or reversed by the presence or supplementation of vitamins D3 and K2.