Inadequate calcium intake or intestinal malabsorption is closely related to osteoporosis. The aim of this study was to investigate the effects of sea cucumber enzymatic hydrolysates (SCEH) prepared from body wall on calcium absorption in calcium-deficient rats. Fifty-six female Sprague-Dawley rats on a low calcium diet that induced osteoporosis were fed SCEH and calcium carbonate for 3 months. The results showed that oral administration of SCEH and calcium carbonate both increased the body weight (bw) and bone mineral density (BMD) in calcium-deficient rats to varying degrees, thereby alleviating the adverse effects of osteoporosis. In addition, oral administration of a combination of SCEH (2000 mg/kg bw/day) and calcium (333 mg/kg bw/day) upregulated the expression of calcium transporter genes (TRPV6 and Calbindin-D9k) in the intestines, increased serum 1,25(OH)2D3 levels and BMD, decreased trabecular bone separation, and inhibited parathyroid hormone (PTH) release in calcium-deficient rats compared to calcium carbonate groups. Thus, this combination promoted calcium absorption and improved the efficiency of calcium utilization. These results might be associated with a higher short chain fatty acid (SCFA) level in the gut and an increased abundance of butyrate and acetate producing bacteria, such as Christensenellaceae R7 group, UCG-005 and Clostridium UCG-014. Therefore, a diet containing SCEH and calcium combinations might be recommended to increase bone mass and inhibit osteoporosis.