The company, whose Chief Executive Officer Michael Saylor is one of the most vocal proponents of bitcoin, bought the cryptocurrency in cash at an average price of about $52,765 each.
MicroStrategy, the world's largest publicly traded business intelligence company, spent last year steadily amassing bitcoin after making its first investment in August.
James said the civil settlement with Hong Kong-based iFinex Inc and related entities will also require them to halt trading activity with New Yorkers.
Tether is the world's third-largest cryptocurrency, after Bitcoin and Ethereum, according to CoinMarketCap. Its market value was about $34.8 billion on Tuesday, up from $2.8 billion when James announced her charges in April 2019.
Bitcoin hit a new high of $50,602, and was last up 5% at $50,300. It has risen around 72% so far this year, with most of the gains coming after electric carmaker Tesla said it had bought $1.5 billion in bitcoin.
It also said it would accept the currency as payment.