Zearalenone (ZEA) is a naturally occurring foodborne mycotoxin and is of widespread interest because of its estrogen-like effects. However, the impact of the myco-estrogen ZEA on bone health remains incompletely understood. This study investigates the mechanisms underlying the effect of ZEA on bone health by establishing osteoporosis and osteoblast models in aged laying hens. ZEA exhibited deleterious effects on hens’ bone health by reducing femoral bone weight and bone mineral density (BMD), decreasing serum calcium (Ca) and phosphorus (P), elevating levels of alkaline phosphatase (ALP) and parathyroid hormone (PTH), diminishing the medullary bone micro-structural parameters (utilizing Micro-CT) and the proportion of trabeculae bone, and down-regulating the expression of osteogenic factors in bone. Interestingly, we found that hBMSCs differentiated towards adipocytes rather than osteoblasts in the ZEA micro-environment, which may be one of the reasons why ZEA causes bone damage. Subsequent experiments involving primary osteoblasts and hFob1.19 cell lines demonstrated that ZEA induced osteoblast apoptosis and impeded osteoblast proliferation. Transcriptome analysis revealed that ZEA primarily hindered osteoblast proliferation by activating the MAPK signaling pathway. Western blot identified that ZEA significantly increased ERK and P38 phosphorylation, with further confirmation through ERK and P38 phosphorylation antagonists. ZEA primarily targets the estrogen receptor (ER), specifically ERα, to activate the ERK/P38 MAPK signaling pathway. In summary, ZEA activates the ERK/P38 MAPK signaling pathway by targeting ERα, thereby inhibiting osteoblast proliferation and promoting osteoclast apoptosis, ultimately leading to decreased bone formation and increased osteoporosis.