Exhaustive exercise is associated with an increase in circulating glucocorticoids (GCs), lymphocyte apoptosis, and a reduction in intestinal lymphocyte number. The present study examined the role of GCs on the numerical changes seen in intestinal lymphocytes after exercise. Female C57BL/6 mice were bilaterally adrenalectomized (ADX; n = 18) or given sham surgery (Sham; n = 18) and assigned to one of three exercise conditions: treadmill running (28 m/min, 90 min, 2° slope) and killed immediately or after 24 h recovery, or not exercised and killed immediately after 90-min exposure to the treadmill environment. Lymphocytes were isolated from the intestines with CD45⁺ cells collected by positive selection using magnetic bead separation columns, and lymphocyte subpopulations were analyzed by flow cytometry for CD45⁺, CD3αβ⁺, CD3γδ⁺, CD8β⁺, CD8α⁺, CD4⁺, and NK⁺ phenotypic markers. ADX mice had significantly more intestinal CD45⁺ leukocytes (P < 0.05) and CD3αβ⁺ (P < 0.05), CD3γδ⁺ (P < 0.01), CD8α⁺ (P < 0.001), and NK⁺ (P < 0.05) intestinal lymphocytes than Sham mice. There was a significant effect of exercise condition on total intestinal CD45⁺ leukocytes (P < 0.01) and CD3αβ⁺ (P < 0.05), CD8α⁺ (P < 0.001), and CD4⁺ (P < 0.05) intestinal lymphocytes, with fewer cells at 24 h postexercise compared with the other treatment conditions. There were no surgical × exercise interaction effects on the CD3 and CD8 phenotype numbers. Plasma corticosterone was virtually nil in ADX mice regardless of exercise condition but was significantly elevated in Sham mice immediately postexercise (P < 0.001). The data indicate that ADX does not prevent the loss of lymphocytes from the intestinal mucosa 24 h after strenuous exercise and GCs are not directly causal in the leukopenia of exercise.