The United States international forward's move to the Britannia Stadium was announced in August and initially blocked in January but, after a loan at Utrecht, it has failed again
The Premier League club announced in August that the 21-year-old had signed a pre-contract agreement to join from the New England Revolution at the start of 2014.
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The United States international joined Utrecht on loan while his Stoke future was being resolved but the Staffordshire side have suffered another blow in a bid to get permission to play the Amercican.
A brief statement on the club's official website on Saturday read: "Stoke City can confirm the club has been unsuccessful in its attempt to secure a work permit for Juan Agudelo.
"It is the second time the club have had an application for a work permit for the 21-year-old United States international rejected by an appeals panel."
Agudelo has scored three goals in 14 Eredivisie appearances for Utrecht this season.