Eight Malians and eight Mauritanians were killed at the Diabaly checkpoint in the Segou region while on their way to a conference in Bamako, Mali's capital, Voice of America reported.
Security officials said soldiers opened fire on the preachers after they failed to obey a command to stop.
Media reports said the preachers were not armed but may have appeared to be terrorists because of their long beards.
Islamic militants took control of northern Mali after a military coup in Bamako in March.
Notable deaths of 2014 [PHOTOS]