Mouse interleukin-3 (mIL-3) is a critical cytokine regulator of myeloid cell differentiation, survival and activation, and consequently this cytokine has become a key reagent for hematological studies in the laboratory. Although bacterial expression has been used for the preparation of recombinant mIL-3 for more than 20 years, the resultant cytokine is known to exhibit poor solubility, be prone to aggregation, and may contain mispaired disulfide bonds. As a result, little structural characterization of mIL-3 has been possible to date. In the present work, we describe a convenient, inexpensive, and scalable protocol for preparing an mIL-3 analog with wild-type bioactivity from Escherichia coli via a simple purification scheme. This analog is typically expressed at >1 mg/l of shaking Super broth culture and, owing to solubility >5 mg/ml, structural studies in solution by nuclear magnetic resonance spectroscopy are feasible for mIL-3 for the first time.