It looks the upload request is not accepted by the server. JFileUpload uses HEAD/POST (multipart) or PUT depending on how you configured it.

Which server-side script are you using? PHP?

Multipart upload with resume support requires HTTP PUT and you've setup JS3Upload for HTTP POST, could you try to replace mode parameter from: jfileupload.transfer.client.s3.rest.POSTUploadTransfer by: jfileupload.transfer.client.s3.rest.PUTUploadTransfer as you can see under applet_s3_multipart.js ...

Did you start from multipart sample provided with JS3Upload?

And what happen if you do not modify this line?
Which PHP version do you have?

1.7.0_55?

Which server-side script are you using? PHP, JSP or ?

Are you under Windows or MacOSX?

Is resume enable in PHP script?

Could you check in Java Control Panel that Java is setup to use a proxy? If it is setup with "No Proxy" then it cannot detect it.