It is an object of the present invention to obtain a soluble thrombomodulin substantially not containing a denatured product of soluble thrombomodulin that may be generated under acidic conditions. The present invention provides a method for producing soluble thrombomodulin substantially not containing a denatured product of the soluble thrombomodulin that may be generated under acidic conditions, from a soluble thrombomodulin-containing material that contains or is suspected to contain the denatured product of the soluble thrombomodulin, which comprises a step of subjecting the soluble thrombomodulin-containing material to an anion exchanger or hydroxyapatite and a step of 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 said gradient is a gradient of salt concentration, 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.
