Temperature-dependent sex determination, where egg incubation temperature irreversibly determines offspring sex, is a common sex-determining mechanism in reptiles. Weather is the primary determinant of temperature in reptile nests, yet the effects of weather are mediated through the nest microhabitat selected by the mother (e.g., overstory canopy cover). One potentially important aspect of the nest microhabitat is the physical substrate used for nesting. However, the influence of substrate type on nest temperature and offspring sex determination has never been experimentally assessed in the field. We incubated eggs of Painted Turtles (Chrysemys picta) in three substrate types similar to those commonly selected for nesting within our study population. Within a single study site, we constructed pits, which we refilled with loam, sand, or gravel. Then, we created artificial nests in each substrate type, and randomly assigned eggs to a substrate treatment. Substrate type influenced nest temperature and soil moisture, and affected incubation duration, but no other phenotype measured beyond offspring sex ratios. The cooler loam yielded the most male-biased outcome. This finding illustrates the potential importance of nesting substrate as a component of nest-site choice and as a factor in modeling future nest temperature scenarios.