John Stith Pemberton was born in Knoxville, Crawford County, Georgia, to James Clifford Pemberton and Martha Patsy Gay. His early life was steeped in the agrarian society of the antebellum South.

Pemberton's childhood involved fundamental education typical for the time, likely receiving instruction at home or in local schools. Details of his early schooling are scarce, but it laid the groundwork for his later pursuit of knowledge in medicine and chemistry.

Pemberton pursued education focused on medicine and pharmacy, showing a strong aptitude for chemistry and related sciences. Precise details and institutions of his medical education are somewhat limited, but historical evidence suggests he received a formal education related to medicine and apothecary studies.

Pemberton earned his license to practice pharmacy. This marked a significant step in his career, equipping him with the credentials and skills necessary to operate a pharmacy and compound medications.