You've probably declared the file as static in app.yaml. Static files are not available to your application; if you need to serve them both as static files and read them as application files, you'll need to include 2 copies in your project (ideally using symlinks, so you don't actually have to maintain an actual copy.)
Update Nov 2014:
As suggested in the comments, you can now do this with the application_readable flag:
application_readable
Optional. By default, files declared in static file handlers are
uploaded as static data and are only served to end users, they cannot
be read by an application. If this field is set to true, the files are
also uploaded as code data so your application can read them. Both
uploads are charged against your code and static data storage resource
quotas.
See https://cloud.google.com/appengine/docs/python/config/appconfig#Static_Directory_Handlers
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