Altered sex hormone levels are thought to play an important role in adult-onset diseases including obesity, cardiovascular disease, and diabetes. They contribute to these complex diseases through changes in their availability, which is influenced, in part, by binding proteins. Insulin resistance, which is characteristic of these diseases, along with increased insulin secretion, is a physiologic change that occurs normally during pregnancy. To determine the relationship between insulin resistance and sex hormone levels, we examined the associations of sex hormone-binding globulin (SHBG) and testosterone with measures of glycemia and insulinemia in a healthy pregnant population. We measured fasting serum SHBG and testosterone levels in 215 Hispanic mothers of Mexican ancestry from the HAPO Study cohort and tested for associations between SHBG and testosterone levels and maternal plasma glucose and C-peptide. After adjusting for confounding variables, serum total testosterone (TT) was positively associated with fasting C-peptide (0.18 μg/l higher for TT higher by 1 SD, p=0.001) and 1-h C-peptide (0.79 μg/l higher for TT higher by 1 SD, p<0.001). Free testosterone (FT) was also positively associated with fasting C-peptide (0.19 μg/l higher for FT higher by 1 SD, p<0.001), and 1-h C-peptide (0.83 μg/l higher for FT higher by 1 SD, p<0.001). Although these findings are from a single cohort, this study provides evidence for an association between testosterone and C-peptide during pregnancy in a nondiabetic Hispanic obstetric population.