The Shanghai-Kunming line -- 2,252 km in length -- traverses the five provinces of Zhejiang, Jiangxi, Hunan, Guizhou and Yunnan and cuts travel time from Shanghai to Kunming from 34 to 11 hours, China Railway Corporation said.
The maximum speed is 330 km per hour, Wang Jinda, a train driver was quoted as saying by the state-run Xinhua news agency.
The line is also the longest east-west high-speed railway in China.
A longer rail line stretching north to south is the 2,298-km Beijing-Guangzhou line, put into operation in 2012. China has built more than 20,000 km of high-speed rail lines.
The launch of the Shanghai-Kunming line means the country's high-speed rail grid has taken shape, connecting almost all provinces on the Chinese mainland.
China is also aggressively marketing its bullet train technology in different countries, including India.