Soybean [Glycine max (L.) Merr.] contains several health promoting compounds including phytosterols, isoflavones, phytic acid, and protease inhibitors. The two abundant protease inhibitors of soybean seeds are the Kunitz trypsin inhibitor and the Bowman-Birk inhibitor (BBI). Bowman-Birk inhibitor has been touted as a potential cancer chemopreventive agent for humans. Little information is available on the effect of growing location on the accumulation of this cancer chemopreventive agent. In this study we have examined the protein profile of eight soybean varieties that were grown in three Missouri locations in 2009 and 2010. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated that soybean varieties that were grown in Grand Pass contained elevated levels of the β subunit of β-conglycinin and reduced amounts of BBI. This observation was further confirmed by Western blot analysis. This difference in the levels of BBI was also reflected in the chymotrypsin inhibitor activity. Growing location also influenced the overall S content of soybean seeds as evidenced by inductively coupled plasma mass spectrometry analysis. Seeds grown in Grand Pass had lower amounts of total S content in both 2009 and 2010. Our results demonstrate that growing location has a profound effect on the accumulation of BBI and it is possible to modulate the concentration of this cancer chemopreventive agent by simple changes in agronomic practices.