A method is provided for producing soluble thrombomodulin substantially not containing a denatured product of the soluble thrombomodulin that is generated under acidic conditions, which comprises: subjecting the soluble thrombomodulin-containing material to an anion exchanger or hydroxyapatite and carrying out linear gradient elution, stepwise gradient elution, or gradient elution in which linear gradient elution is combined with stepwise gradient elution under separation conditions in which the denatured product of the soluble thrombomodulin can be separated, wherein the gradient is a salt concentration gradient, so as to obtain an elution fraction containing soluble thrombomodulin that does not substantially contain the denatured product of the soluble thrombomodulin, either (a) after the position of a fraction has previously been confirmed, or (b) while confirming the elution fraction.
