The attack on the border with Egypt was the first suicide bombing targeting Hamas forces in Gaza, security sources said.
While the border remained open after the bombing, Hamas police carried out raids on Salafist extremists, arresting dozens, witnesses and a security source said.
The explosion struck at around 1:00 am near the Gaza Strip's lone crossing with Egypt along the Sinai Peninsula, where radical Islamists are waging an insurgency against Egyptian forces.
The second man was moderately wounded, while the four other guards were also wounded, including one seriously, the sources said.
Eyewitnesses said hundreds of security forces deployed along the border after the explosion.
Hamas's military wing, the Ezzedine al-Qassam Brigades, said field commander Nidal al-Jaafari, 28, was killed in the attack.
Qassam posted a series of photos of Jaafari in military fatigues carrying different weapons.
The group blamed "fundamentalist jihadists" for the attack, with the attacker later named as 20-year-old Jamal Kallab from Gaza.
Hamas, itself an Islamist movement, has run the Gaza Strip since 2007 but has been regularly criticised by more radical Salafist groups in the impoverished, Israeli-blockaded coastal territory.
They have often claimed responsibility for firing rockets at Israel from inside Gaza, sparking military responses from the Jewish state.
There have been threats of retaliation in recent months over arrests by Hamas, according to security sources in Gaza.
After the explosion, Hamas security forces arrested dozens of alleged Salafists, a Hamas security source said.
Salafist groups claim thousands of supporters in the Palestinian enclave of some two million people, while Hamas estimates they number only in the dozens.
The Hamas source said initial indications were that the two men involved in today's attack were acting alone, without coordination with Salafi leaders.
After an emergency meeting between Hamas and Gaza's other political factions, a statement said they "completely reject the bombing in Rafah and stress that the incident benefits the (Israeli) occupation".
Hamas has recently boosted its forces along the border with Egypt as it seeks to improve relations with Cairo.
Egypt has kept the Rafah crossing mostly closed in recent years, though it opened it on Monday for four days to allow Muslims to travel to Mecca in Saudi Arabia for the hajj pilgrimage, as well as for some humanitarian cases.
The border crossing stayed open today but was due to be closed from tomorrow.
Egypt and Israel are the only countries bordering Gaza.
Israel has maintained a decade-long blockade on the strip it says is necessary to prevent Hamas from obtaining weapons or materials that could be used to manufacture arms.
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