This study was conducted to enhance broccoli secondary metabolites; using plant biotechnology techniques and tissue culture as well as to evaluate the therapeutic effect of broccoli extract against osteoporosis in ovariectomized (OVX) rats. Cell culture technique was implemented in order to achieve and produce polyphenols from leaf explants of broccoli. The high-performance liquid chromatography (HPLC) technique was used to evaluate the polyphenolic profiles of ethanolic extracts for cell cultures compared to the dried inflorescences of broccoli (cultivated plant). However, total phenol and flavonoid contents were determined by spectrophotometric technique. Induction of osteoporosis was performed via ovarectomy operation where rats were divided into 5 groups (10 rats each). Group 1: Sham operated control group and the remaining rats were ovariectomized for 12 weeks to produce osteoporosis model. Group 2: Ovariectomized osteoporotic positive control group. Groups 3-5: Ovariectomized rat daily received 17‐β‐estradiol (10 µg/kg), broccoli cell suspension culture ethanol extracts (300 & 600 mg/kg) orally. The obtained results showed that Murashige and Skoog basal nutrient medium (MS) fortified with 2.25 µM of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.55 µM of kinetin (kin.) showed the best medium for calli production. Addition of methionine enhanced polyphenolic production in cell suspension culture which was identified by HPLC. Administration of broccoli cell suspension extract elevated the uterine weight, high density lipoprotein-cholesterol and estradiol 2 (E2) serum levels and reduced serum levels of lipid profile, liver and kidney functions, osteocalcin and total procollagen type 1 N-terminal propeptide (TPINP). In conclusion, broccoli may be a promising sources of secondary metabolites; polyphenol contents and flavonoids for treatment of bone loss and cell damage in osteoporosis.