Ten of the people found in the sweltering trailer died.
James Matthew Bradley Jr., 60, faces five charges, including one count of conspiracy to transport and harbor undocumented aliens for financial gain resulting in death and one count of transportation of undocumented aliens resulting in death.
If convicted of those charges, he could be sentenced to the death penalty or up to life in prison.
Bradley has said he was not aware of the cargo in his vehicle and only discovered it when he parked at a Walmart in San Antonio and went outside to urinate.
One of Bradley's public defenders, Kimberly Stevens, told CNN she could not comment on an ongoing case.
Eight people were found dead in the trailer, and two died at hospitals. Of the other 29 people discovered, two are still hospitalized. Others have been charged as material witnesses.
Material witness laws allow officials to detain those who may have knowledge of crimes committed by others to ensure they will testify in legal proceedings, including grand jury sessions and trials.
Bradley is in federal custody and the case will be heard in the western district of Texas. One of the other charges against him is possession of a firearm by a felon.
A news release from prosecutors didn't say when Bradley will next appear in court.
The tractor-trailer was found parked at a Walmart in San Antonio on July 23 and an employee at the store called police. Bradley told police he was driving from Laredo to San Antonio. According to a criminal complaint filed in July, Bradley also said the trailer refrigeration system didn't work.
Temperatures in San Antonio that weekend reached 100 degrees.