In 1577, the Franciscan missionaries arrived in Manila, and in 1578 they started evangelizing Laguna, Morong (now Rizal), Tayabas (now Quezon) and the Bicol Peninsula. Juan de Plasencia and Diego de Oropesa were the earliest Franciscans sent to these places. From 1580, the towns of Bay, Caliraya, Majayjay, Nagcarlán, Liliw, Pila, Santa Cruz, Lumban, Pángil and Siniloan were founded. In 1678, Fray Hernando Cabrera founded San Pablo de los Montes (now San Pablo City) and built a wooden church and convent considered as the best and finest in the province.
In 1670, delimitation of borders were made between Lucban, Majayjay and Cavite. The populous town at that time was Bay, the capital of the province until 1688, when the seat of the provincial government was moved to Pagsanján, and later in 1858, to Santa Cruz. In 1754, the Province of Laguna and Tayabas were divided, with the Malinao River separating the towns of Majayjay and Lucbán.
The province became a bloody battle ground for the Chinese during the two instances that they rose in revolt against Spain. In 1603s, the Chinese made their last stand in the mountains of San Pablo, and in 1639, they fortified themselves in the highlands of Cavinti and Lumban, surrendering in Pagsanjan a year later.
The natives of Laguna proved loyal to the Spanish crown during the British invasion (1762–1764) when thousands rallied to its defense. When a detachment of British troops under Captain Thomas Backhouse entered the province in search of the silver cargo of the galleon Filipino, Francisco de San Juan of Pagsanján led a band of volunteers that fought them in several engagements in and around the town which was then the provincial capital (1688–1858). Backhouse plundered the town and burned its newly reconstructed church but San Juan succeeded in escaping with the precious hoard to Pampanga where the treasure greatly bolstered the defense effort of Simón de Anda, leader of the resistance movement. For his heroism, San Juan was made a brigade commander and alcalde mayor of Tayabas (now Quezon) province.
The people's loyalty gradually degenerated into bitter hostility. Grave abuses by the colonizers, especially those of the clergy, caused the resentment of the natives to be fanned into a rising flood of insurrection. In 1840 for instance, religious intolerance led the people of Majayjay, Nagcarlan, Bay, and Biñan to join the revolt of Hermano Pule (Apolinario de la Cruz) of Lucban, Tayabas.
Laguna was also exposed to the aspirations of its most famous son, Dr. José Rizal, who was born in Calamba City. The persecution of the Rizal family, along with their fellow landowners toward the end of the century further aggravated the situation. In 1896, thousands of inhabitants, especially of Bay, Los Baños, Nagcarlan, Magdalena, Santa Cruz, and Pagsanjan had joined the revolutionary Katipunan.
