The teat canal-one of the primary defense mechanisms of the udder-ensures the milk ow during milking in bovines and prevents pathogens from entering the udder by forming a barrier through the elastic muscle and keratin layers tightly closing the surrounding area. The current study investigated the effects of blood calcium status on teat closure in cows after milking. The study covered 200 healthy teats, of which 100 were from normocalcemic (NC) cows and 100 were from subclinical hypocalcemic (SCH) cows. Teat canal length (TCL) and width (TCW) were measured with ultrasonography at 0-min premilking and 15-and 30-mins post-milking. Cylindrically shaped teat canal volume (TCV) was calculated by deriving from TCL and TCW. Time-dependent changes in teat canal closure and their relationships with blood calcium levels were analyzed. The results showed that the calcium level did not affect TCL, TCW, and TCV (P > 0.05) during the 15 minutes post-milking period. However, TCL (P < 0.001), TCW (P < 0.05), and TCV (P < 0.001) were higher in NC cows than in SCH cows at 30 minutes post-milking. At 15 minutes post-milking, no correlation existed between the teat canal closure (ΔTCL, ΔTCW, and ΔTCV) and the blood calcium level, while signi cant correlations were available between the teat canal closure and the blood calcium level {ΔTCL (r:-0.288, P < 0.001), ΔTCW (r:-0.260, P < 0.001), ΔTCV (r:-0.150, P < 0.05)} at 30 minutes post-milking. The current study concluded that the blood calcium status signi cantly impacts the teat canal closure in bovines, and calcium status should be meticulously monitored with the mastitis control program to apply necessary strategic steps.