The use of non-drug intervention for calcium deficiency has attracted attention in recent years. Although calcium carbonate is the preferred raw material for calcium supplementation, the mechanism of chondroitin sulfate, a drug for the treatment of osteoarthritis, in alleviating osteoporosis from the perspective of intestinal flora and metabolomics, has been rarely reported. In this study, intestinal microflora abundance and fecal and plasma metabolite expression levels were compared in control rats fed a basal diet, rats fed a low-calcium diet, rats fed a low-calcium diet plus calcium carbonate, and rats fed a low-calcium diet plus chondroitin sulfate. The results showed that calcium content and bone mineral density in the midpoint and distal femur in the calcium carbonate and chondroitin sulfate groups were significantly increased compared with the low-calcium group. 16s rRNA sequencing and metabolomics analysis showed that chondroitin sulfate intervention changed the intestinal microflora composition and fecal and plasma metabolic profiles of low-calcium fed rats, and promoted calcium absorption and alleviated osteoporosis through anti-inflammatory and antioxidant mechanisms. Correlation analysis showed that the selected intestinal flora was significantly correlated with metabolites enriched in feces and plasma. In conclusion, chondroitin sulfate intervention may promote calcium absorption, alleviate the symptoms of osteoporosis in low-calcium rats, and affect intestinal microflora composition and metabolic characteristics. This study provides scientific evidence of the potential impact of chondroitin sulfate as a palliative agent or dietary supplement for patients with osteoporosis.