Osteoporosis is a global bone metabolic disease of diverse etiology and complex pathology, with high incidence and disability, in which insufficient osteogenesis plays a crucial role. Due to the obvious curative effects, natural herbal ingredients have attracted more and more attentions. Kaempferol, a kind of flavonoids, can significantly improve osteogenesis. However, the detailed mechanism remains unclear. Hence, we established osteoporotic models in vitro and in vivo, utilizing serum-starved MC3T3-E1 cells and orchidectomized rats, to investigate the mechanism and overall efficacy of kaempferol. In vitro, kaempferol directly promoted the proliferation and differentiation of serum-starvation-treated MC3T3-E1 cells. Additionally, quantitative chemoproteomic profiling combined with cellular interference assays confirmed that kaempferol could activate Pik3r1. The pro-proliferation of kaempferol on MC3T3-E1 was eliminated after knockdown of Pik3r1, conversely, overexpression of Pik3r1 led to a stronger pro-proliferative effect. Furthermore, treatment of kaempferol elevated damaged bone mineral density and ameliorated bone histomorphometry indexes in osteoporotic rats, including increased bone volume fraction, trabecular thickness and number, decreased trabecular separation and structural model index, and improved skeletal sequences. Summarily, kaempferol was effective in increasing bone mineral density and improving bone morphology as well as structure in castrated rats, and promoted osteogenesis of MC3T3-E1 via activating Pik3r1. Our work was a useful attempt to investigate the anti-osteoporotic mechanism of natural herbal ingredients and will provide new insights into the treatment strategy for osteoporosis.