Osteoblast uses aerobic glycolysis to meet the metabolic needs in differentiation process. Lactate, the end product of glycolysis, presents in the environment with elevated PTH and osteoblast differentiation. Although previous findings showed that lactate promoted osteoblast differentiation, whether lactate affects PTH mediated osteoblast differentiation is unclear. To investigate this, pre-osteoblast cell line MC3T3-E1 was treated PTH with or without physiological dose of lactate. Lactate increases ALP positive cell formation, increases ALP activity and expression of differentiation related markers, enriches the CREB transcriptional factor target genes in PTH treated cells. Using inhibitors for MCT-1 reveales that lactate effects are MCT-1 independent. Lactate selectively increases Akt and p38 activation but not Erk1/2 and beta-Catenin activation. The inhibitors for Akt and p38 inhibit lactate effects on PTH mediated osteoblast differentiation. Using inhibitors for G alpha i signaling of GPR81 further increases Alp mRNA levels in lactate and PTH co-treatment cells. However, with the inhibitors for G beta gamma-PLC-PKC signaling, the effect of lactate on PTH mediated osteoblast differentiation is inhibited. Our data demonstrate that lactate activates GPR81-G beta gamma-PLC-PKC-Akt signaling to regulate osteoblast differentiation that mediated by PTH treatment. (C) 2018 Elsevier Inc. All rights reserved.