Follow the set up instructions for Mach3 as the software needs to know how far the axis has moved. The microstepping is for getting increased resolution not calibrating the axis movement. Here is the link to download the install and configuration guide. Page 5-23 covers the calibration for a given movement. However you should also check that you have the motors set up properly as set out in the rest of chapter 5.
ArtSoft USA - Documentation
There are other documents on that page that you may find helpful and there are some good video instructions for download at ArtSoft USA - Video Tutorials
Hope this gets you going - Regards - Pat