Whitworth developed great skill as a mechanic while working for Maudslay, developing various precision machine tools and also introducing a box casting scheme for the iron frames of machine tools that simultaneously increased their rigidity and reduced their weight.
Whitworth also worked for Holtzapffel & Co (makers of ornamental lathes) and Joseph Clement. While at Clement's workshop he helped with the manufacture of Charles Babbage's calculating machine, the Difference engine. He returned to Openshaw, Manchester, in 1833 to start his own business manufacturing lathes and other machine tools, which became renowned for their high standard of workmanship. In 1850, architect Edward Walters was commissioned to build The Firs for Whitworth. This was a grand mansion at Fallowfield Manchester, which still stands today, functioning as Chancellors Hotel & Conference Centre.
His next innovation, in 1840, was a measuring technique called "end measurements" that used a precision flat plane and measuring screw, both of his own invention. The system, with a precision of one millionth of an inch, was demonstrated at the Great Exhibition of 1851.
In 1841 Whitworth devised a standard for screw threads with a fixed thread angle of 55° and having a standard pitch for a given diameter. This soon became the first nationally standardized system; its adoption by the railway companies, who until then had all used different screw threads, leading to its widespread acceptance. It later became a British Standard, "British Standard Whitworth", abbreviated to BSW and governed by BS 84:1956.
The Enfield rifle was converted to Snider-Enfield Rifle by Jacob Snider, a Dutch-American wine merchant from Philadelphia. By converting existing Enfield rifles this way, the cost of a "new" breech-loading Snider-Enfield rifle was only 12 shillings.
Queen Victoria opened the first meeting of the British Rifle Association at Wimbledon, in 1860 by firing a Whitworth rifle from a fixed mechanical rest. The rifle scored a bull's eye at a range of 400 yards (366 m).
While trying to increase the bursting strength of his gun barrels, Whitworth patented a process called "fluid-compressed steel" for casting steel under pressure, and built a new steel works near Manchester. Some of his castings were shown at the Great Exhibition in Paris ca. 1883.
A strong believer in the value of technical education, Whitworth backed the new Mechanics' Institute in Manchester, which was to become UMIST, and helped found the Manchester School of Design. In 1868, he founded a scholarship for the advancement of mechanical engineering. In recognition of his achievements and contributions to education in Manchester, the Whitworth Building of the University of Manchester's Main Campus is named in his honour, as well as the University Halls of residence "Whitworth Park" and one of the main streets in Manchester's city centre, "Whitworth Street".
Whitworth died in Monte Carlo, where he had travelled in the hope of improving his health. He was buried at the church of Darley (or Darley Dale) St Helen in Derbyshire. A detailed obituary was published in the American magazine The Manufacturer and Builder (Volume 19, Issue 6, June 1887). He directed his trustees to spend his fortune on philanthropic projects, which they still do to this day.