Spermatozoa experience a long and tough transit in male and female genital tracts before successful fertilization. Glycosylation helps spermatogenesis, epididymal maturation, passing through cervical mucus, avoiding killing of the female immunologic system, and shaking hands between sperm and egg. Changes in glycosylations along the transit ensure that the right things happen at the right time and place on spermatozoa. Aberrant glycosylations on spermatozoa will negatively affect their fertility. Thus, we developed a lectin array method to examine the glycocalyx of spermatozoa, which will help observe glycosylations occurring on spermatozoa in a normal or abnormal conditions, such as spermatozoa with DEF126 mutation and poor freezability. Intriguingly, binding levels of ABA (Agaricus bisporus agglutinin), a lectin marking the inner layer of the glycocalyx, were changed in these subfertile spermatozoa, which indicates that the integrity of glycocalyx is critical for sperm fertility. In this chapter, we reviewed the impacts of glycosylations on sperm fertility, the lectin array method, and its potential application for sperm function assessment.