The motion was defeated by 122 votes to 97 in the 234-member House.
Rebel AIADMK camp leader O Panneerselvam and his 10 MLAs were not present during the voting.
Earlier, Deputy Speaker Pollachi V Jayaraman went for voice vote twice and declared that the motion had been defeated but the DMK insisted on a division which Jayaraman permitted.
DMK and its allies cast 97 votes in favour of the motion while 122 AIADMK MLAs, including Mylapore MLA R Natraj who had supported the rebels during the trust vote last month, voted against it.
After the motion was defeated today, Dhanapal resumed his chair and asserted that he was always 'impartial'.
While Leader of the House K A Sengottaiyan hailed Dhanapal for 'giving more opportunity' to opposition members to speak, DMK Deputy Leader Duraimurugan said they moved the motion expecting him to win appreciation of the opposition as well.
Earlier, moving the motion, Leader of Opposition M K Stalin referred to various happenings in the House since the AIADMK assumed power last May and alleged that the Speaker had been favouring the ruling party.
The motion was seconded by Duraimurugan and supported by allies the Congress and the IUML.
Criticising Dhanapal for his conduct on February 18 when the trust vote was taken up, Stalin claimed the Speaker refused to realise that secret ballot was a "reasonable" demand.
Stalin said while the Supreme Court had directed secret ballot in states like Uttar Pradesh, Dhanapal "did not come forward to use his powers" to hold the trust vote in a "fair manner".
(REOPENS MDS8)
Stalin said Dhanapal declined to expunge "defamatory," remarks against his party while he ordered removal of remarks against ruling party.
"He gave opportunity to ruling party members to air defamatory views outside on subjects debated in the House," Stalin alleged.
Referring to deployment of police personnel and enmasse eviction of his party members on that day, he said DMK MLAs suffered injuries in the melee and the Speaker conducted the voting proceedings with only ruling party members present in the House.
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