The effect of exercise training on hematopoietic stem cells (HSC) is largely unknown. The aim of the present investigation was to determine whether exercise training could expand the bone marrow HSC pool and influence various aspects of HSC function. Mice were either exercise trained (EX; 1 h/day, 3 days/wk, for 8 wk) or remained sedentary (SED). Bone marrow (BM) from SED or EX mice was extracted from different HSC niches for cell cycle analysis, HSC (lineage(-), Sca-1(+), c-Kit(+)) quantification, and differentiation along various hematopoietic lineages via flow cytometry. Serum was collected for evaluation of cytokines known to regulate HSC. To determine HSC function, BM from EX and SED mice was transplanted into primary and secondary recipients in a BM transplant assay. EX increased HSC quantity in the vascular BM niche 20% vs. SED (P < 0.05) and increased the proportion of whole BM cells in G(2)/M phase of cell cycle (P < 0.05). The number of spleen colonies was 48% greater (P < 0.05) in recipients transplanted with BM from EX. Serum IL-6 levels were decreased 38% in EX, and differentiation along the lineage trended to increase (16%, P = 0.053 and 16%, P = 0.061, respectively). Short- or long-term engraftment and homing in primary recipients were not altered in EX. HSC self-renewal as analyzed by hematopoietic regeneration in secondary recipients was also unaffected by EX. Here we demonstrate that HSC quantity is increased in the BM niche associated with more activated, differentiated HSC, and that this expansion does not improve or impair HSC function.