We examined spermatozoa from 135 male patients consulting for infertility to evaluate the usefulness of the hypo-osmotic swelling (HOS) test of sperm fertility for the swim-up washing method. HOS test values significantly improved following the treatment of spermatozoa using the swim-up washing method. There were no differences in the rate of sperm motility between the normal group and the oligozoospermic group following treatment, but the HOS test identified significant differences in swelling rates in both groups before and after treatment. The results of the HOS test following swim-up washing were higher during fertilization attempts for 11 cases of successful intra-uterine insemination (IUI) than during unsuccessful IUI attempts; there were no significant differences between the control group and the successful IUI cases. Moreover, using the lower-normal limits for overall sperm swelling (52%) and g-type sperm swelling (30%) obtained from a normal control group, we found that swelling rates were higher when IUI was successful. These findings indicate that the HOS test is an effective measure of sperm fertilizing function when the swim-up washing method is used for sperm treatment. The score of g-type sperm swelling can be used as a substitute for overall sperm swelling.