Spiders from Laos collected on expeditions from 2003 to 2008 are investigated. Currently, 89 spider species are recorded from Laos. Records of 51 species are included in the present paper. Four species are described for the first time: Argiope dang sp. n. (male, female) from Vang Vieng, Pseudopoda wang sp. n. (male, female) from Nam Khan valley, Olios muang sp. n. (male) from Ban Thathot, and Thelcticopis folia sp. n. (female) from Nam Khan valley. Sixteen species are recorded from Laos for the first time: Acusilas malaccensis Murphy & Murphy 1983, Agelena limbata Thorell 1897, Anepsion maritatum (O. Pickard-Cambridge 1877, Arachnura melanura Simon 1867, Araneus ellipticus (Tikader & Bal 1981), Argiope pulchella Thorell 1881, Eriovixia pseudocentrodes (Bösenberg & Strand 1906), Hersilia flagellifera Baehr & Baehr 1993, Menemerus bivittatus (Dufour 1831), Myrmarachne turriformis Badcock 1918, Oxyopes birmanicus Thorell 1887, Portia assamensis Wanless 1978, Tetragnatha ceylonica O. Pickard-Cambridge 1869, Tetragnatha lauta Yaginuma 1959, Tylorida striata (Thorell 1877), Tylorida ventralis (Thorell 1877). New observations on the biology of Eurychoera banna Zhang, Zhu & Song 2004 are documented. E. quadrimaculata Thorell 1897 is firstly recorded from Brunei. The following species are transferred from the genus Zygiella (Araneidae) to the genus Guizygiella (Tetragnathidae): Guizygiella indica (Tikader & Bal 1980) comb. nov., Guizygiella shivui (Patel & Reddy 1990) comb. nov. Illustrations are given for Argiope pulchella, A. ?versicolor, Eriovixia pseudocentrodes, Laoponia saetosa Platnick & Jägerger 2008, Orsinome vethi (Hasselt 1882), Oxyopes birmanicus, Tylorida ventralis, mostly showing intraspecific variation. A map of Laotian collecting localities is given. Orsinome vethi is recorded for Flores the first time, the record represents the eastern most distribution of this species.