Hundreds marched to the city police station carrying signs saying "Stop killing us" and "Resistance is beautiful," but the atmosphere was far calmer than the previous two nights.
Pressure was growing on police to release video of the shooting of Keith Lamont Scott, a 43-year-old African American, whose killing on Tuesday sparked the unrest.
Scott's death was the latest in a string of police- involved killings of black men that have fueled outrage across the United States.
An overnight curfew was also in place.
"We are going to be a lot more proactive," Charlotte police chief Kerr Putney told a news conference. "We made 44 arrests last night because we are not going to tolerate the behavior."
A protestor shot by a civilian in Wednesday night's protests died in hospital yesterday, local media reported.
Scott was shot and killed in an apartment complex parking lot on Tuesday during an encounter with police officers searching for another person wanted for arrest.
The authorities have so far refused to release police video of the incident.
However, members of Scott's family watched the footage yesterday, raising "more questions than answers," their lawyers said.
No gun is visible in the video, which shows Scott stepping backward when he was shot, one of the lawyers told CNN.
"His hands are down by his side. He is acting calm," Justin Bamberg said. "You do see something in his hand, but it's impossible to make out from the video what it is."
The video footage "does not give me absolute definitive visual evidence that would confirm that a person is pointing a gun," he told CNN.
But the footage indicates the officer identified as having shot Scott -- Brentley Vinson, who is also black -- was justified, he added.
A handful of protesters confronted police last night. However, many marched past officers who posed a less intimidating presence on the streets despite their greater numbers.
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