Thirteen-lined ground squirrels are strictly diurnal, coming above ground when the sun is high and the earth is warm, and returning to the warmth and safety of their burrows long before sundown. They rarely venture out of the burrow on damp, dark, or overcast days. When they venture out, they will often stand upright, with front paws held close to the chest, surveying their domain. If danger threatens, they run, with tail held horizontally, to the nearest burrow. The inconcpicuous 2-inch (5cm) diameter burrow opening is often concealed by vegetation and rarely has soil scattered in front of it like a woodchuck's burrow. The main entrance plunges down 6 inches (15 cm) or more before angling off into a complex system of galleries and side entranceways. The nesting chamber, about 9 inches (23 cm) in diameter and lined with fine dry grass, is located somewhat deeper than the main burrow system. The thirteen-lined ground squirrel's natural enemies include just about all predators, especially hawks, badgers, weasels, foxes, coyotes, bull snakes, and black snakes.

Thirteen-lined ground squirrels begin hibernation in September or early October and emerge between late March and early May in the northern portions of their range. In southern Texas, they have been observed above ground as late as October 27 and as early as January. Males usually begin hibernation earlier in the fall and emerge earlier in the spring than females. When they hibernate, their body temperature is generally 3 degrees C of the ambient air temperature. When active, their body temperature can vary 8 to 10 degrees C, without ill effect.

Mating activity begins within 2 weeks after the squirrels emerge from hibernation. Both sexes are sexually active for about 2 weeks. After a gestation period of 28 days, 3 to 14 (average 10) blind, naked, and toothless young are born. Only 1 litter is produced per year. Young ground squirrels weigh about 1/10th ounce (3 to 4 g) at birth. Their stripes begin to appear after about 12 days and their eyes open 28 to 30 days after birth. Young squirrels are weaned and on their own after 6 to 12 weeks. Thirteen-lined ground squirrels are sexually mature at 9 or 10 months of age.

The thirteen-lined ground squirrel’s preference for insects and field mice may provide some benefit to the agricultural community. Large concentrations of these ground squirrels in pastures, fields, and garden can, however, cause loss of forages and crops. They dig up newly planted seeds, clip emerging plant shoots, and pull over-ripening wheat, barley, and oats to eat the grain. They will readily feed on commonly grown home or truck garden vegetables, often damaging much more than they consume.

Thirteen-lined ground squirrels will invade golf courses, parks, lawns, athletic fields, cemeteries, and similar wide open grassy sites, their burrowing and feeding activity can cause major economic and aesthetic damage in such places.

Ground squirrels can either be live-trapped, using a squirrel bait and then relocated, or they can be gassed in their burrows using "Giant Destroyers".