The objective of this study was to investigate the effects of dietary fenugreek seed powder (FSP) supplementation in layers. A total of 288 layers (26 weeks old, Hyline-brown) were divided into 3 groups fed a corn-soybean meal diet (corn 50.4%, soybean meal 18.7%) containing 0 [control (CON)], 0.05 or 0.1% FSP for 5 weeks. Data were statistically analyzed via ANOVA using the GLM procedure of SAS for a completely randomized design, replicated 8 times with 12 layers per replication. Egg production was recorded daily on a hen-day basis, and egg quality and blood profiles were analyzed using an egg multi tester and an automatic biochemistry blood analyzer, respectively. For the measurement of cecal Lactobacillus and E. coli, cecal digesta was serially diluted in 0.1% peptone water, and incubated onto agar plates at 37°C for 24 h, under anaerobic and aerobic conditions, respectively. A Gastec gas sampling pump was used to detect gases in layer excreta. The inclusion of FSP in the layer diet did not affect egg production, feed intake and feed conversion among treatments. However, egg weight (CON = 63.1 vs. 0.1% FSP = 66.4 g), eggshell breaking strength (CON = 4.39 vs. 0.1% FSP = 4.66 kg/cm²), eggshell thickness (CON = 35.4 vs. 0.1% FSP = 36.7 mm⁻²) and yolk color (CON = 7.0 vs. 0.1% FSP = 7.8) was increased in FSP-fed groups (P < 0.05). Supplemental FSP decreased serum total cholesterol concentration (CON = 222 vs. 0.05% FSP = 172, 0.1% FSP = 184 mg/dL), whereas the HDL-cholesterol (CON = 33 vs. 0.1% = FSP 44 mg/dL) and IgG concentration (CON = 446 vs. 0.1% FSP = 469 mg/dL) was increased in the FSP fed-groups (P < 0.05). The inclusion of FSP led to an increase in cecal Lactobacillus number (CON = 6.98 vs. 0.1% FSP = 7.42 cfu/g; P < 0.05) and a decrease in E. coli number (CON = 6.57 vs. 0.05% FSP = 6.14 cfu/g; P < 0.05) and excreta ammonia gas emission (CON = 48 vs. 0.1% FSP = 30 ppm; P < 0.05). These results suggest that the addition of FSP does not increase egg production, but may affect egg quality, serum total- and HDL-cholesterol, IgG concentration and cecal microflora. The inclusion of FSP also decreased ammonia gas emission in layer excreta.