The sale netted Costolo over $5 million. He still owns more than 800,000 shares in the company, according to the filing.
In December, two of Costolo's family trusts liquidated the remainder of their shares in the social media company, for roughly $20 million.
Though the stock is up slightly since the beginning of the year, it has fallen dramatically since its highs in February 2014. The stock's decline, plus a widely shared sentiment that the company is strategically rudderless, has caused a number of analysts to demand Costolo's exit. (Nevertheless, he still has his supporters.)
A spokesman for Twitter said in an email: "Dick has sold shares under a plan filed in the summer and his total sales represent less than 10% of his total equity in Twitter."
Twitter shares were around $38 a share in early trading Wednesday.
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