In 1899 Barkla was admitted to Trinity College, Cambridge, with an 1851 Research Fellowship from the Royal Commission for the Exhibition of 1851, to work in the Cavendish Laboratory under the Physicist J. J. Thomson (discoverer of the electron). During his first two years at Cambridge, Barkla would, under the directions of Thomson, study the velocity of electromagnetic waves along wires of different widths and materials.
After a year and a half at Trinity College, Cambridge, his love of music led him to transfer to King's College, Cambridge, in order to sing in their chapel choir. Barkla's baritone voice was of remarkable beauty and his solo performances would always be fully attended. He completed his Bachelor of Arts degree in 1903, and then his Master of Arts degree in 1907. He married Mary Esther Cowell in the same year, with whom he would have two sons and one daughter.
In 1913, after having worked at the Universities of Cambridge, Liverpool, and King's College London, Barkla was appointed as a Professor of Natural Philosophy at the University of Edinburgh in 1913, a position that he held until his death.
Barkla made significant progress in developing and refining the laws of X-ray scattering, X-ray spectroscopy, the principles governing the transmission of X-rays through matter, and especially the principles of the excitation of secondary X-rays. For his discovery of the characteristic X-rays of elements, Barkla was awarded the Nobel Prize in Physics in 1917. He was also awarded the Hughes Medal of the British Royal Society that same year.
The lunar crater Barkla was named in the honour of Charles Barkla. A commemorative plaque has been installed in the vicinity of the Canongate, near the Faculty of Education Buildings, at the University of Edinburgh. Additionally, a lecture theatre at the University of Liverpool's Physics department, as well as a Biophysics laboratory in the Biological science department, are named after him. In 2012 a gritter in Barkla's home town of Widnes was named in his honour, following a competition run by the local newspaper.