Glyphosate (N-phosphonomethylglycine) is a widely used organophosphorus herbicide that inhibits the shikimate pathway, a crucial metabolic route responsible for the synthesis of aromatic amino acids in plants and certain microorganisms. Due to its broad-spectrum activity, glyphosate serves as the main active ingredient in various commercial herbicide formulations, including Roundup and Kalach 360 SL (KL). It poses a health hazard to animals and humans due to its persistence in soil, water erosion, and crops. The aim of our study was to continue the previous research to explore the impact of KL on bone using physico-chemical parameters and in silico studies after exposing female rats for 60 days. The in silico study concerned the assessment of binding affinity and molecular interactions using computational modeling approach. The rats were allocated into three experimental groups: group 1 ( = 6) served as controls, while groups 2 and 3 received low and high doses (Dose 1: 126 mg/Kg and Dose 2: 315 mg/Kg) of KL dissolved in water, respectively. All rats were sacrificed after 60 days of exposure. XRD and FTIR spectrum analysis of bone tissues in female rats showed significant histoarchitectural changes associated with bone mineralization disruption. Our results have demonstrated that sub-chronic exposure of adult female rats to KL causes bone rarefaction, as confirmed by a previous histological study. This physico-chemical study has further confirmed the harmful impact of KL on the crystalline fraction of bone tissue, composed of hydroxyapatite crystals. In addition, the computational analyses showed that glyphosate binds to 3 Glu form of osteocalcin (3 Glu-OCN) (4MZZ) and decarboxylated osteocalcin (8I75) with good affinities and strong molecular interactions, which justified and supported the in vivo findings. In conclusion, KL may interfere with hydroxyapatite and osteocalcin and, therefore, impair bone remodeling and metabolism.